Oligoribonucleotides and methods of use thereof for treatment of fibrotic conditions and other diseases

ABSTRACT

The invention relates to a double-stranded compound, preferably an oligoribonucleotide (siRNA), which down-regulates the expression of a human TGaseII gene at the post-transcriptional level. The invention also relates to a pharmaceutical composition comprising the compound, or a vector capable of expressing the oligoribonucleotide compound, and a pharmaceutically acceptable carrier. The present invention also contemplates a method of treating a patient suffering from a fibrotic disease such as pulmonary, kidney and liver fibrosis or ocular, scarring comprising administering to the patient the pharmaceutical composition in a therapeutically effective dose so as to thereby treat the patient. The invention also relates to treatment of fibrotic and other diseases by use of antibodies to TGaseII polypeptide.

This application claims priority of U.S. Provisional patent applicationsNo. 60/540,687, filed Jan. 30, 2004 and No. 60/641,522, filed Jan. 4,2005, both of which are hereby incorporated by reference in theirentirety.

Throughout this application various patent and scientific publicationsare cited. The disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

siRNAs and RNA interference

RNA interference (RNAi) is a phenomenon involving double-stranded (ds)RNA-dependent gene specific posttranscriptional silencing. Originally,attempts to study this phenomenon and to manipulate mammalian cellsexperimentally were frustrated by an active, non-specific antiviraldefense mechanism which was activated in response to long dsRNAmolecules; see Gil et al. 2000, Apoptosis, 5:107-114. Later it wasdiscovered that synthetic duplexes of 21 nucleotide RNAs could mediategene specific RNAi in mammalian cells, without the stimulation of thegeneric antiviral defence mechanisms see Elbashir et al. Nature 2001,411:494-498 and Caplen et al. Proc Natl Acad Sci 2001, 98:9742-9747. Asa result, small interfering RNAs (siRNAs), which are shortdouble-stranded RNAs, have become powerful tools in attempting tounderstand gene

Thus, RNA interference (RNAi) refers to the process of sequence-specificpost-transcriptional gene silencing in mammals mediated by smallinterfering RNAs (siRNAs) (Fire et al, 1998, Nature 391, 806) ormicroRNAs (miRNAs) (Ambros V. Nature 431:7006,350-355(2004); and BartelD P. Cell. 2004 Jan. 23; 116(2): 281-97 MicroRNAs. genomics, biogenesis,mechanism, and function). The corresponding process in plants iscommonly referred to as specific post-transcriptional gene silencing orRNA silencing and is also referred to as quelling in fungi. An siRNA isa double-stranded RNA molecule which down-regulates or silences(prevents) the expression of a gene/mRNA of its endogenous (cellular)counterpart. RNA interference is based on the ability of dsRNA speciesto enter a specific protein complex, where it is then targeted to thecomplementary cellular RNA and specifically degrades it. Thus, the RNAinterference response features an endonuclease complex containing ansiRNA, commonly referred to as an RNA-induced silencing complex (RISC),which mediates cleavage of single-stranded RNA having a sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA may take place in the middle of the region complementaryto the antisense strand of the siRNA duplex (Elbashir et al 2001, GenesDev., 15, 188). In more detail, longer dsRNAs are digested into short(17-29 bp) dsRNA fragments (also referred to as short inhibitoryPNAs—“siRNAs”) by type III RNAses (DICER, DROSHA, etc., Bernstein etal., Nature, 2001, v.409, p.363-6; Lee et al., Nature, 2003, 425,p.415-9). The RISC protein complex recognizes these fragments andcomplementary mRNA. The whole process is culminated by endonucleasecleavage of target mRNA (McManus&Sharp, Nature Rev Genet , 2002, v.3,p.737-47; Paddison &Hannon, Curr Opin Mol Ther. 2003 June; 5(3):217-24). For information on these terms and proposed mechanisms, seeBernstein E., Denli A M. Hannon G J: 2001 The rest is silence. RNA. I;7(11): 1509-21; Nishikura K.: 2001 A short primer on RNAi. RNA-directedRNA polymerase acts as a key catalyst. Cell. I 16; 107(4): 415-8 and PCTpublication WO 01/36646 (Glover et al).

The selection and synthesis of siRNA corresponding to known genes hasbeen widely reported; see for example Chalk A M, Wahlestedt C,Sonnhammer E L. 2004 Improved and automated prediction of effectivesiRNA Biochem. Biophys. Res. Commun. Jun. 18; 319(1): 264-74; Sioud M,Leirdal M., 2004, Potential design rules and enzymatic synthesis ofsiRNAs, Methods Mol Biol.; 252:457-69; Levenkova N, Gu Q, Rux J. J. 2004Gene specific siRNA selector Bioinformatics. I 12; 20(3): 430-2. andUi-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A,Ueda R, Saigo K., Guidelines for the selection of highly effective siRNAsequences for mammalian and chick RNA interference Nucleic Acids Res.2004 I 9;32(3):936-48.Se also Liu Y, Braasch D A, Nulf C J, Corey D R.Efficient and isoform-selective inhibition of cellular gene expressionby peptide nucleic acids, Biochemistry, 2004 I 24;43(7):1921-7. See alsoPCT publications WO 2004/015107 (Atugen) and WO 02/44321 (Tuschl et al),and also Chiu Y L, Rana T M. siRNA function in RNAi. a chemicalmodification analysis, RNA 2003 September;9(9):1034-48 and I PatentNos.5898031 and 6107094 (Crooke) for production of modified/ more stablesiRNAs.

Several groups have described the development of DNA-based vectorscapable of generating siRNA within cells. The method generally involvestranscription of short hairpin RNAs that are efficiently processed toform siRNAs within cells. Paddison et al. PNAS 2002, 99:1443-1448;Paddison et al. Genes & Dev 2002, 16:948-958; Sui et al. PNAS 2002,8:5515-5520; and Brummelkamp et al. Science 2002, 296:550-553. Thesereports describe methods to generate siRNAs capable of specificallytargeting numerous endogenously and exogenously expressed genes.

siRNA has recently been successfully used for inhibition in primates;for further details see Tolentino et al., Retina 24(1) February 2004 1132-138.

Transglutaminase (TGase) Family

Transglutaminases (EC 2.3.2.13) are a family of enzymes that catalyzethe crosslinking of proteins by epsilon-gamma glutamyl lysine isopeptidebonds. The family comprises 9 different enzymes among which are thefactor XIIIa (plasma transglutaminase), keratinocyte transglutaminase(TGasel), epidermal transglutaminase (TGaseIII), prostatetransglutaminase (TGaseIV), and tissue-type transglutaminase (TGaseII).Although the overall primary structure of these enzymes is different,they all share a common amino acid sequence at the active site(Y-G-Q-C-W) and a strict calcium dependence for their activity (LesortM, Tucholski J, Miller M L, Johnson G V, Tissue transglutaminase: apossible role in neurodegenerative diseases. Prog Neurobiol. 2000August; 61(5):439-63).

Transglutaminase II. Transglutaminase II (TGaseII) also known asProtein-glutamine gamma-glutamyltransferase, TGase C, TGC, TG(C), andtissue-type transglutaminase, is a unique multifunctional enzyme withdual enzymatic activity:

-   I) The enzyme acts as a TGase protein (Ca²⁺ activated,    GTP-inactivated) with crosslinking activities (i.e. it catalyzes    reactions resulting in protein cross-links and/or covalent    incorporation of biogenic amines). TGase further catalyzes the    formation of a covalent glutamyl—lysyl bond, a unique isopeptide    bond that is highly resistant to proteolysis and denaturants and    that cannot be disrupted by any known vertebrate endopeptidase.-   2) The enzyme also acts as a GTP-binding protein that transduces the    activating signal from alpha 1 B and alpha 1 D adrenergic receptors,    from TP alpha thromboxane A2 receptor and from oxytocin receptor to    phospholipase C delta 1 (i.e. it activates inositol phosphate    production, Ca²⁺ mobilization etc.). It has been shown that both    alpha ID adrenergic receptor and PLC act as guanine nucleotide    exchanging factor for transglutaminase 11 (Baek K J, Kang S, Damron    D, Im M, Phospholipase C-delta1 is a guanine nucleotide exchanging    factor for transglutaminase II (G alpha h) and promotes alpha    1B-adrenoreceptor-mediated GTP binding and intracellular calcium    release. J Biol Chem. 2001 Feb. 23;276(8):5591-7).

The dual function of this enzyme is separate and the active sites arelocated at different positions (Im M. J, Russell M A, Feng J F,Transglutaminase II. a new class of GTP-binding protein with newbiological functions. Cell Signal. 1997 November;9(7):477-82), andinhibition of crosslinking activity of TGaseII may be achieved withoutthe interference with its G-protein function.

TGaseII: Subcellular Localization and Fibrosis Related IntracellularSignaling Activity

TgaseII is ubiquitously expressed and can be found in association withECM and intracellularly, both as membrane bound and as cytosolicprotein. The GTP-binding activity is higher in the membrane fraction ofTGase and the cross-linking activity is higher in the cytosolic andextracellular fraction. On the cell surface, TGase binds to fibronectinvia its 42 kDa gelatin-binding domain. The cross-linking of fibronectinwith collagen contributes to structural stabilization of the ECMrendering the matrix resistant to proteolysis by matrixmettaloproteinases. Thus TGaseII, by favoring deposition ofextracellular proteins and inhibiting breakdown of said proteins,contributes to ECM accumulation, a phenomenon that is central to theformation of tissue fibrosis.

In addition, TGase mediates the binding of latent TGF-beta to the ECM, arequired step for a proper subsequent processing of this pro-fibroticfactor, which finally results in the release of active TGF-beta (Le etal., Connect Tissue Res. 2001;42(4):245-53. Rosenthal et al., ArthritisRheum. 2000 August;43(8): 1729-33).

In the cytoplasm, TGaseII may activate RhoA, a small G protein of theras family known to have an important role in cytoskeletalrearrangement, regulation of cell morphology and differentiation.Activation of TGaseII results in an increased transamidation of RhoA,which then functions as a constitutively active G-protein showingincreased binding to and activation of its downstream target ROCK-2(Rho-associated kinase) (Singh et al. EMBO J. 2001 May15;20(10):2413-23). The specific ROCK kinase inhibitor, Y-27632, showedanti-fibrotic effect in UUO model of renal fibrosis as well as in themodels of liver or lung fibrosis (Nagatoya K, Moriyama T, Kawada N,Takeji M, Oseto S, Murozono T, Ando A, Imai E, Hori M. Y-27632 was foundto prevent tubulointerstitial fibrosis in mouse kidneys with unilateralurethral obstruction. Kidney Int. 2002 May;61(5):1684-95; Murata T, AriiS, Nakamura T, Mori A, Kaido T, Furuyama H, Furumoto K, Nakao T, IsobeN, Imamura M, Inhibitory effect of Y-27632, a ROCK inhibitor, onprogression of rat liver fibrosis in association with inactivation ofhepatic stellate cells. J Hepatol. 2001 October;35(4):474-81; Shimizu Y,Dobashi K, lizuka K, Horie T, Suzuki K, Tukagoshi H, Nakazawa T,Nakazato Y, Mori M, Contribution of small GTPase Rho and its targetprotein ROCK in a murine model of lung fibrosis. Am J Respir Crit CareMed. 2001 January;163(1):210-7). Recently, a unique function of TGaseIIcontributing to activation of NFkB, a transcription factor with awell-documented pro-fibrotic activity was described (Baud et al., J SocBiol. 2002;196(4):269-73, Lee at al. J Biol Chem. 2004 Dec.17;279(51):53725-35).

Fibrotic Diseases

Fibrotic diseases are all characterized by the excess deposition of afibrous material within the extracellular matrix, which contributes toabnormal changes in tissue architecture and interferes with normal organfunction. Unfortunately, although fibrosis is widely prevalent,debilitating and often life threatening, there is no effective treatmentcurrently available.

All tissues damaged by trauma respond by the initiation of awound-healing program. Fibrosis, a type of disorder characterized byexcessive scarring, occurs when the normal self-limiting process ofwound healing response is disturbed, and causes excessive production anddeposition of collagen. As a result, normal organ tissue is replacedwith scar tissue, which eventually leads to the functional failure ofthe organ.

Fibrosis may be initiated by diverse causes and in various organs. Livercirrhosis, pulmonary fibrosis, sarcoidosis, keloids and kidney fibrosisare all chronic conditions associated with progressive fibrosis, therebycausing a continuous loss of normal tissue function.

Acute fibrosis (usually with a sudden and severe onset and of shortduration) occurs as a common response to various forms of traumaincluding accidental injuries (particularly injuries to the spine andcentral nervous system), infections, surgery, ischemic illness (e.g.cardiac scarring following heart attack), burns, environmentalpollutants, alcohol and other types of toxins, acute respiratorydistress syndrome, radiation and chemotherapy treatments).

For further information on different types of fibrosis see: Molina V,Blank M, Shoenfeld Y. (2002), “Fibrotic diseases”, Harefuah, 141(11):973-8, 1009; Yu L, Noble NA, Border WA (2002), “Therapeutic strategiesto halt renal fibrosis”, Curr Opin Pharmacol. 2(2):177-81; Keane W F,Lyle P A. (2003), “Recent advances in management of type 2 diabetes andnephropathy: lessons from the RENAAL study”, Am J Kidney Dis. 41(3 Suppl2): S22-5; Bohle A, Kressel G, Muller C A, Muller G A. (1989), “Thepathogenesis of chronic renal failure”, Pathol Res Pract. 185(4):421-40;Kikkawa R, Togawa M, Isono M, Isshiki K, Haneda M. (1997), “Mechanism ofthe progression of diabetic nephropathy to renal failure”, Kidney IntSuppl. 62:S39-40; Bataller R, Brenner D A. (2001), “Hepatic stellatecells as a target for the treatment of liver fibrosis”, Semin Liver Dis.21(3):437-51; Gross T J, Hunninghake G W, (2001) “Idiopathic pulmonaryfibrosis”, N Engl J Med. 345(7):517-25; Frohlich E D. (2001) “Fibrosisand ischemia: the real risks in hypertensive heart disease”, Am JHypertens;14(6 Pt 2):194S-199S.

Liver Fibrosis

Liver fibrosis (LF) is a generally irreversible consequence of hepaticdamage of several etiologies. In the Western world, the main etiologiccategories are: alcoholic liver disease (30-50%), viral hepatitis (30%),biliary disease (5-10%), primary hemochromatosis (5%), and drug-relatedand cryptogenic cirrhosis of unknown etiology(10-15%). Wilson's disease,α₁-antitrypsin deficiency and other rare diseases also have liverfibrosis as one of the symptoms Scheinberg IH, Sternlieb I., Wilsondisease and idiopathic copper toxicosis. Am J Clin Nutr 1996May;63(5):842S-5S; Parfrey H, Mahadeva R, Lomas D A.,Alpha(I)-antitrypsin deficiency, liver disease and emphysema. Int JBiochem Cell Biol. 2003 July;35(7):1009-14.

Liver cirrhosis, the end stage of liver fibrosis, frequently requiresliver transplantation and is among the top ten causes of death in theWestern world.

Anti-inflammatory agents, which cause inhibition of activation ofhepatic stellate cells, stimulation of growth of hepatocytes andinhibition of post translational modification of collagen have all beenused to treat liver fibrosis. However, due to the lack of selectivetargeting, these treatments suffer from, inter alia, the drawbacks ofsevere side effects,.

For more information see Friedman S L. (2003), “Liver fibrosis—frombench to bedside”, J Hepatol. 38 Suppl 1:S38-53; Albanis E, Safadi R,Friedman S L. (2003), “Treatment of hepatic fibrosis: almost there”,Curr Gastroenterol Rep. 5(1):48-56. See also Grenard P, Bresson-Hadni S,El Alaoui S, Chevallier M, Vuitton D A, Ricard-Blum S.,Transglutaminase-mediated cross-linking is involved in the stabilizationof extracellular matrix in human liver fibrosis. J Hepatol. 2001September;35(3):367-75 ;Mirza A, Liu S L, Frizell E, Zhu J, Maddukuri S,Martinez J, Davies P, Schwarting R, Norton P, Zern M A., A role fortissue transglutaminase in hepatic injury and fibrogenesis, and itsregulation by NF-kappaB. Am J Physiol. 1997 February;272(2 Pt I ):G281-8).

Kidney Fibrosis and Related Conditions

Chronic Renal Failure (CRF)

Chronic renal failure is a gradual and progressive loss of the abilityof the kidneys to excrete wastes, concentrate urine, and conserveelectrolytes. CRF is slowly progressive. It most often results from anydisease that causes gradual loss of kidney function, and fibrosis is themain pathology that produces CRF.

Diabetic Nephropathy

Diabetic nephropathy, hallmarks of which are glomerulosclerosis andtubulointerstitial fibrosis, is the single most prevalent cause ofend-stage renal disease in the modern world, and diabetic patientsconstitute the largest population on dialysis. Such therapy is costlyand far from optimal. Transplantation offers a better outcome butsuffers from a severe shortage of donors. More targeted therapiesagainst diabetic nephropathy (as well as against other types of kidneypathologies) are not developed, since molecular mechanisms underlyingthese pathologies are largely unknown. Identification of an essentialfunctional target gene that is modulated in the disease and affects theseverity of the outcome of diabetes nephropathy has a high diagnostic aswell as therapeutic value.

Origins of kidney Pathology

Many pathological processes in the kidney ( e.g., glomerular nephritis,physical obstructions, toxic injuries, metabolic and immunologicaldiseases) eventually culminate in similar or identical morphologicalchanges, namely glomerulosclerosis and tubulointerstitial fibrosis.Thus, different types of insults converge on the same single geneticprogram resulting in two hallmarks of fibrosis: the proliferation offibroblasts and overproduction by them of various protein components ofconnective tissue. In addition, thickening of the basal membrane in theglomeruli accompanies interstitial fibrosis and culminates inglomerulosclerosis. See also Johnson TS, Skill NJ, El Nahas A M, OldroydS D, Thomas G L, Douthwaite J A, Haylor J L, Griffin M, Transglutaminasetranscription and antigen translocation in experimental renal scarring.J Am Soc Nephrol. 1999 October;10(10):2146-57; Johnson T S, Griffin M,Thomas G L, Skill J, Cox A, Yang B, Nicholas B, Birckbichler P J,Muchaneta-Kubara C, Meguid El Nahas A, The role of transglutaminase inthe rat subtotal nephrectomy model of renal fibrosis. J Clin Invest.1997 Jun. 15;99(12):2950-60).

Pulmonary Fibrosis

Interstitial pulmonary fibrosis (IPF) is scarring of the lung caused bya variety of inhaled agents including mineral particles, organic dusts,and oxidant gases, or by unknown reasons (idiopathic lung fibrosis). Thedisease afflicts millions of individuals worldwide, and there are noeffective therapeutic approaches. A major reason for the lack of usefultreatments is that few of the molecular mechanisms of disease have beendefined sufficiently to design appropriate targets for therapy (Lasky .J A., Brody A R. (2000), “Interstitial fibrosis and growth factors”,Environ Health Perspect.; 108 Suppl 4:751-62).

The cellular substrate of pathogenesis of pulmonary fibrosis includesendothelial and epithelial cell injury, production of inflammatory cellsand their mediators, and fibroblast activation., Fibrosis is believed tobe related to a dysregulation in cross-talk between inflammatory andstructural cells, mediated by various cytokines, chemokines and growthfactors, which are responsible for the maintenance of tissue homeostasisand which coordinate the response to injury (Kelly M, Kolb M, BonniaudP, Gauldie J. (2003), “Re-evaluation of fibrogenic cytokines in lungfibrosis“Curr Pharm Des. 9(1):39-49).

Conventional therapy consisting of glucocorticoids or cytotoxic drugs isusually ineffective in preventing progression of the disease. It isbelieved that further understanding of the molecular mechanisms ofendothelial and epithelial cell injury, inflammatory reaction,fibroblast proliferation, collagen deposition and lung repair, isnecessary for the development of effective treatments against pulmonaryfibrosis (Kuwano K, Hagimoto N, Hara N. (2001), “Molecular mechanisms ofpulmonary fibrosis and current treatment”, Curr Mol Med. 1(5):551-73).See also Griffin M, Smith L L, Wynne J., Changes in transglutaminaseactivity in an experimental model of pulmonary fibrosis induced byParaquat. Br. J. Exp. Pathol. 1979 December; 60(6):653-61).

Cardiac Fibrosis

Heart failure is unique among the major cardiovascular disorders in thatit alone is increasing in prevalence while there has been a strikingdecrease in other conditions. Some of this can be attributed to theaging of the populations of the United States and Europe. The ability tosalvage patients with myocardial damage is also a major factor, as thesepatients may develop progression of left ventricular dysfunction due todeleterious remodelling of the heart.

The normal myocardium is composed of a variety of cells, cardiacmyocytes and noncardiomyocytes, which include endothelial and vascularsmooth muscle cells and fibroblasts. (Weber KT. (2000), “Fibrosis andhypertensive heart disease”, Curr Opin Cardiol. 15(4):264-72).

Structural remodeling of the ventricular wall is a key determinant ofclinical outcome in heart disease. Such remodeling involves theproduction and destruction of extracellular matrix proteins, cellproliferation and migration, and apoptotic and necrotic cell death.Cardiac fibroblasts are crucially involved in these processes, producinggrowth factors and cytokines that act as autocrine and paracrinefactors, as well as extracellular matrix proteins and proteinases.Recent studies have shown that the interactions between cardiacfibroblasts and cardiomyocytes are essential for the progression ofcardiac remodeling of which the net effect is deterioration in cardiacfunction and the onset of heart failure (Manabe I, Shindo T, Nagai R.(2002), “Gene expression in fibroblasts and fibrosis: involvement incardiac hypertrophy”, Circ Res. 13;91(12):1103-13).

The use of agents to block the renin-angiotensin-aldosterone andsympathetic nervous systems has been shown to inhibit (and sometimeseven reverse) cardiac remodelling and to improve the clinical course ofpatients with cardiac dysfunction. However, drugs aiming at directinhibition or reduction of fibrosis are not yet available. See alsoGreenberg B. (2001), “Treatment of heart failure: state of the art andprospectives”, J Cardiovasc Pharmacol. 38 Suppl 2:S59-63, Zhang Z, VezzaR, Plappert T, McNamara P, Lawson J A, Austin S, Pratico D, Sutton M S,FitzGerald G A, COX-2-dependent cardiac failure in Gh/tTG transgenicmice. Circ Res. 2003 May 30;92(10):1153-61. Epub 2003 Apr. 17; Hwang KC, Gray C D, Sweet W E, Moravec C S, Im M J., Alpha I-adrenergicreceptor coupling with Gh in the failing human heart. Circulation. 1996Aug. 15;94(4):718-26.

Neurological Diseases

Polyglutamine diseases are a group of neurological diseases that arecaused by expansion of CAG trinucleotide repeats coding forpolyglutamine insert. Polyglutamine diseases include Huntington'sdisease (HD), spinobulbar muscular atrophy, dentatorubral-pallidoluysianatrophy and spinocerebellar ataxias (SCAs) 1, 2, 3, 6, 7 and 17. Allthese diseases are characterized by the presence of expansion ofpolyglutamine stretches (exceeding 35-40 glutamines), thus formingintranuclear aggregates, which leads to neuronal death. Alzheimer'sdisease (AD) is the most common cause of cognitive impairment in olderpatients and is expected to increase greatly in prevalence.Neurofibrillary degeneration, associated with the formation of pairedhelical filaments (PHF), is one of the critical neuropathologicalhallmarks of Alzheimer's disease (AD). Parkinson disease is aneurodegenerative disorder of aging characterized by a selective andprogressive loss of dopaminergic neurons within the substantia nigra.See also Mastroberardino P G, Iannicola C, Nardacci R, Bernassola F, DeLaurenzi V, Melino G, Moreno S, Pavone F, Oliverio S, Fesus L,Piacentini M. Tissue transglutaminase ablation reduces neuronal deathand prolongs survival in a mouse model of Huntington's disease. CellDeath Differ. 2002 September;9(9):873-80; Karpuj M V, Becher M W,Springer .I E, Chabas D, Youssef S, Pedotti R, Mitchell D, Steinman L.,Prolonged survival and decreased abnormal movements in transgenic modelof Huntington disease, with administration of the transglutaminaseinhibitor cystamine. Nat Med. 2002 February;8(2):143-9; Citron B A, SuoZ, SantaCruz K, Davies P J, Qin F, Festoff B W., Protein crosslinking,tissue transglutaminase, alternative splicing and neurodegeneration.Neurochem Int. 2002 January;40(1):69-78; Chen J S, Mehta K., Tissuetransglutaminase: an enzyme with a split personality. Int J Biochem CellBiol. 1999 Aug.;31(8):817-36.

Osteoarthritis

Among the main characteristics of osteoarthritis are the degradation ofarticular cartilage and the formation of new bone at the joint edges,so-called osteophytes. See Van den Berg W B., Growth factors inexperimental osteoarthritis: transforming growth factor beta pathogenic?J Rheumatol Suppl. 1995 February;43.143-5; Scharstuhl A, Glansbeek H L,Van Beuningen H M, Vitters E L, Van der Kraan P M:, Van den Berg W B.,Inhibition of endogenous TGF-beta during experimental osteoarthritisprevents osteophyte formation and impairs cartilage repair. J Immunol.2002 Jul. 1; 169(1):507-14; Karpouzas G A, Terkeltaub R A., Newdevelopments in the pathogenesis of articular cartilage calcification.Curr Rheumatol Rep. 1999 Dec; 1(2):121-7.

Ocular Diseases

Age-related cataracts: Cataracts are characterized by aggregation andcovalent cross-linking of the crystallins, the major structural proteinsof the eye lens, resulting in increase light scattering, opacificationand cataract. Disturbance of calcium homeostasis in the tissue is one ofthe factors implicated in cataractogenesis. see Shridas et al., FEBSLett. 2001 Jun. 22;499(3):245-50; Shin et al, J Biol Chem. 2004 Apr.9;279(15):15032-9; Wan et al., Br J Ophthalmol. 2002November;86(11):1293-8. and Takeuchi N, Kamei A. Biol Pharm Bull. 2000March;23(3):283-90.

Proliferative vitreoretinopathy Proliferative vitreoretinopathy (PVR) isthe most common complication following retinal detachment and associatedwith a retinal hole or break. PVR refers to the growth of cellularmembranes within the vitreous cavity and on the front and back surfacesof the retina containing retinal pigment epithelial (RPE) cells. Thesemembranes, which are essentially scar tissues, exert traction on theretina and may result in recurrences of retinal detachment, even afteran initially successful retinal detachment procedure.

Migration and adhesion of dislocated retinal pigment epithelial (RPE)cells to a fibronectin-rich extracellular matrix is an initial step inproliferative vitreoretinopathy (PVR). See also Casaroli-Marano R P etal Invest Ophthalmol Vis Sci. 1999 August;40(9):2062-72). and PriglingerS et al, Invest Ophthalmol Vis Sci. 2004 March;45(3):955-63; Priglingeret al., Invest Ophthalmol Vis Sci. 2003 January;44(1):355-64.

Fibrosis following glaucoma filtering operation. The goal of theglaucoma filtration procedure is to create a new passageway by whichaqueous fluid inside the eye can escape, thereby lowering the pressure.The filter, therefore, allows the drainage of fluid from inside theanterior chamber of the eye to a “pocket” created between theconjuctiva, which is the outermost covering of the eye, and the sclera,which is the underlying white anatomical structure of the eye. The fluidis eventually absorbed by blood vessels. Unfortunately, due topostoperational scarring, the generated drainage system may be blockedrequiring additional surgical intervention(s).

Current anti-scarring regimens (Mitomycin C or 5FU) are limited due tothe complications involved (e.g. blindness) e.g. see Cordeiro M F, Gay JA, Khaw P T., Human anti-transforming factor-beta2 antibody: a newglaucoma anti-scarring agent Invest Ophthalmol Vis Sci. 1999September;40(10):2225-34.

In conclusion, there are no effective modes of therapy for the diseasesdescribed above, and there is a need, therefore, to develop noveleffective compounds and methods of treatment for these purposes.

SUMMARY OF THE INVENTION

The invention provides novel double stranded oligoribonucleotides. Theseoligoribonucleotides inhibit human TGaseII via the mechanism of RNAinterference. The invention also provides a pharmaceutical compositioncomprising such an oligoribonucleotide, and a vector capable ofexpressing the oligoribonucleotide. The present invention also providesa method of treating a patient suffering from a fibrosis-relatedpathology comprising administering to the patient theoligoribonucleotide typically as a pharmaceutical composition, in atherapeutically effective dose so as to thereby treat the patient. Thepresent invention also contemplates treating other diseases andconditions. The invention also relates to treatment of fibrotic andother diseases by use of an antibody to TGaseII polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. This figure sets forth the nucleotide sequence of the humanTGaseII cDNA-(gi|13653650|ref|XM 009482.3|-ORF)-SEQ ID NO:1.

FIG. 2. This figure sets forth the amino acid sequence of the humanTGaseII corresponding polypeptide—SEQ ID NO:2.

FIG. 3. Western Blot results demonstrating the effect of various TGaseIIsiRNAs on human TGaseII polypeptide expression.

The figure demonstrates the activity of various siRNAs in reducingexpression of human TGase polypeptide in HeLa cells, 72 hr followingsiRNA transfection. GAPDH expression serves as control for proteinloading. The numbers 15 and 30 represent concentration of theoligonucleotide in nM. “Cells” (first lane in each panel) standsfor—nontransfected control cells. The last lane in each panel containsprotein extracts from HeLa cells transfected with non-relevant (NR)siRNA, to ensure that the decrease in TGaseII expression is specific tothe anti TGase siRNAs activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for treatment of variouspathologies, as recited below, in a subject in need of such treatmentwhich comprises: administering to the subject an amount of an inhibitorof TGaseII polypeptide sufficient to effect a substantial inhibition ofthe TGaseII activity so as to thereby treat the subject. The TGaseIIinhibitor may be L683685, an antibody to TGaseII polypeptide or an siRNAto TGaseII RNA or any of the TGaseII inhibitors known in the art (forfurther information on such inhibitors see for example: U.S. Pat. No.5,021,440; 4,968,713 and 5,098,707). In particular the present inventionrelates to specific siRNAs targeting TGaseII RNA and the use thereof.The use of neutralizing antibodies against TGaseII is also disclosed.The present invention relates generally to compounds which down-regulateexpression of the human TGaseII gene particularly to novel smallinterfering RNAs (siRNAs), and to the use of these novel siRNAs in thetreatment of various diseases and medical conditions in particularfibrotic diseases, diseases related to fibrotic scarring and otherdiseases related to aberrant expression of Transglutaminase II.

The present invention provides methods and compositions for inhibitingexpression of the target TGaseII gene in vivo. In general, the methodincludes administering oligoribonucleotides, such as small interferingRNAs (i.e., siRNAs) that are targeted to a particular mRNA and hybridizeto, or interact with, it under biological conditions (within the cell),or a nucleic acid material that can produce siRNA in a cell, in anamount sufficient to down-regulate expression of a target gene by an RNAinterference mechanism. In particular, the subject method can be used toinhibit expression of the TGaseII gene for treatment of disease.

Thus, the inhibitor of TGaseII expression (transcription or translation)or polypeptide activity may be inner alia siRNA, antibodies, preferablyneutralizing antibodies or fragments thereof, including single chainantibodies, antisense oligonucleotides, antisense DNA or RNA molecules,proteins, polypeptides and peptides including peptido-mimetics anddominant negatives, and also expression vectors expressing all theabove. Additional inhibitors may be small chemical molecules, whichgenerally have a molecular weight of less than 2000 daltons, morepreferably less than 1000 daltons, even more preferably less than 500daltons. These inhibitors may act as follows: small molecules may affectexpression and/or activity; antibodies may affect activity; all kinds ofantisense may affect TGaseII expression; and dominant negativepolypeptides and peptidomimetics may affect activity; expression vectorsmay be used inter alia for delivery of antisense or dominant-negativepolypeptides or antibodies.

In accordance with the present invention, the siRNA molecules orinhibitors of Transglutaminase II, such as antibodies, may be used asdrugs to treat various pathologies including fibrosis relatedpathologies (as defined below) and also to treat ocular diseasesincluding cataract, cardiovascular diseases, neurological diseases,polyglutamine diseases (including Huntington's disease (HD), spinobulbarmuscular atrophy, dentatorubral-pallidoluysian atrophy andspinocerebellar ataxias (SCAs) 1, 2, 3, 6, 7 and 17), Alzheimer's andParkinson's disease and osteoarthritis.

As used herein, the term “Transglutaminase II gene ”, or “TGaseII gene”,or “TGase gene” is defined as any homolog of TGaseII gene havingpreferably 90% homology, more preferably 95% homology, and even morepreferably 98% homology to the amino acid encoding region of SEQ IDNO:1, or nucleic acid sequences which bind to the TGaseII gene underconditions of highly stringent hybridization, which are well-known inthe art (for example, see Ausubel et al., Current Protocols in MolecularBiology, John Wiley and Sons, Baltimore, Md. (1988), updated in 1995 and1998).

As used herein, the term “Transglutaminase II polypeptide ”, or “TGaseIIpolypeptide”, or “TGase” is defined as any homolog of TGaseIIpolypeptide having preferably 90% homology, more preferably 95%homology, and even more preferably 98% homology to SEQ ID NO:2, aseither full-length or fragments or a domain thereof, as a mutant of thepolypeptide encoded by a spliced variant nucleic acid sequence, as achimera with other polypeptides, provided that any of the above has thesame or substantially the same biological function as the TGaseIIpolypeptide. TGaseII polypeptide, or a TGaseII polypeptide homolog, maybe present in different forms, including but not limited to solubleprotein, membrane-bound (either in purified membrane preparations or ona cell surface), bead-bound, or any other form presenting TGaseIIprotein or fragments and polypeptides derived thereof.

As used herein, an “interactor” is a molecule with which TGaseII bindsor interacts or activates in nature; for example, a molecule on thesurface of a TGaseII polypeptide expressing cell, a molecule on thesurface of a second cell or a cytoplasmic molecule. An interactor may bea ligand that is activated by TGaseII alone or by TGaseII as part of acomplex with other components. An interactor may be a component of asignal transduction pathway that facilitates transduction of anextracellular signal from TGaseII through the cell membrane and into thecell. An interactor, for example, can be a second intercellular proteinthat mediates downstream signaling from TGaseII. The interactor is amolecule with which TGase binds in competition with a known TGasesubstrate (e.g. fibronectin).

As used herein, the term “lysyl donor” or “K donor” is defined as anypolypeptide having the ability to donate a lysyl side chain to allow theformation of gamma-glutamyl-lysine bonds during transglutaminationprocess.

As used herein, the term “glutamyl donor” or “Q donor” is defined as anypolypeptide having the ability to donate glutamine side chain to allowthe formation of gamma-glutamyl-lysine bonds during transglutaminationprocess.

The present invention provides double-stranded oligoribonucleotides(siRNAs), which down-regulate the expression of TGaseII. Thedownregulation of the expression of transglutaminase II can be measuredby e.g., measuring the amount of the lysyl-glutamyl crosslinked materialproduced in the presence of the siRNAs or by direct assessment of theamounts of TGaseII mRNA or polypeptide. The amount of TGaseII mRNA maybe measured by e.g., by Northern blotting, RNase protection, RT-PCR orreal-time PCR. The amount of TGaseII polypeptide may be measured byimmunoblotting or by immunoprecipitation or by ELISA withTGaseII-specific antibodies.

An siRNA of the invention is a duplex oligoribonucleotide in which thesense strand is derived From the mRNA sequence of gene TGaseII, and theantisense strand is complementary to the sense strand. In general, somedeviation from the target mRNA sequence is tolerated withoutcompromising the siRNA activity (see e.g. Czauderna et al 2003 NucleicAcids Research 31(11), 2705-2716). An siRNA of the invention inhibitsgene expression on a post-transcriptional level with or withoutdestroying the mRNA. Without being bound by theory, siRNA may target themRNA for specific cleavage and degradation and/or may inhibittranslation from the targeted message.

There are at least two variant TGaseII polypeptides, for which theGeneBank references are variant 2 NM_(—)198951.1 GI:39777598 and variant1NM_(—)004613.2 GI:39777596. The sequence given in FIG. 1 is thenucleotide sequence of variant 2 (GI:39777598). Both variants and anyother similar minor variants are included in the definition of TGaseIIpolypeptide and in the definition of the TGaseII genes encoding them.

As used herein, the term ” TGaseII gene” is defined as thenaturally-occurring human gene including any allelic variant thereof aswell as any homolog of the TGaseII gene having preferably 90%, homology,more preferably 95% homology, and even more preferably 98% homology tothe amino acid encoding region of SEQ ID NO: 1 or nucleic acid sequenceswhich bind to the TGaseII gene under conditions of highly stringenthybridization, which are well-known in the art (for example, see Ausubelet al., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1988), updated in 1995 and 1998.

As used herein, the term “TGaseII ”, or “TGaseII polypeptide” is definedas the naturally-occurring polypeptide product of the gene including anyallelic variant thereof as well as any homolog of the TGaseIIpolypeptide having preferably 90% homology, more preferably 95%homology, and even more preferably 98% homology to SEQ ID NO:2, aseither full-length or a fragment or a domain thereof, as a mutant or thepolypeptide encoded by a spliced variant nucleic acid sequence, as achimera with other polypeptides, provided that any of the above has thesame or substantially the same biological function as the TGaseIIpolypeptide. More particularly, the invention provides a compound havingthe structure:5′(N)_(x) −Z 3′ (antisense strand)3′Z−(N′)_(y)5′ (sense strand)

-   -   wherein each N and N′ is a ribonucleotide which may be modified        or unmodified in its sugar residue and (N)_(x) and (N′ )_(y) is        oligomer in which each consecutive N or N′ is joined to the next        N or N′ by a covalent bond;    -   wherein each of x and y is an integer between 19 and 40;    -   wherein each of Z and Z′ may be present or absent, but if        present is dTdT and is covalently attached at the 3′ terminus of        the strand in which it is present;    -   and wherein the sequence of (N)_(x) comprises any one of the        antisense sequences present in Tables A, B and C

It will be readily understood by those skilled in the art that thecompounds of the present invention consist of a plurality of nucleotideswhich are linked through covalent linkages. Each Such covalent linkagemay be a phosphodiester linkage, a phosphothioate linkage, or acombination of both, along the length of the nucleotide sequence of theindividual strand. Other possible backbone modifications are describedinter alia in U.S. Pat. Nos. 5,587,361; 6,242,589; 6,277,967; 6,326,358;5,399,676; 5,489,677; and 5,596,086.

In particular embodiments, x and y are preferably an integer betweenabout 19 to about 27, most preferably from about 19 to about 23. In aparticular embodiment of the compound of the invention, x may be equalto y (viz., x=y) and in preferred embodiments x=y=19 or x=y=21. In aparticularly preferred embodiment x=y=19.

In one embodiment of the compound of the invention, Z and Z′ are bothabsent; in another embodiment one of Z or Z′ is present.

In one embodiment of the compound of the invention, all of theribonucleotides of the compound are unmodified in their sugar residues.

In some embodiments of the compound of the invention, at least oneribonucleotide is modified in its sugar residue, preferably amodification at the 2′ position. The modification at the 2′ positionresults in the presence of a moiety which is preferably selected fromthe group comprising amino, fluoro, methoxy, alkoxy and alkyl groups. Ina presently most preferred embodiment the moiety at the 2′ position ismethoxy (2′-O-methyl).

In some embodiments of the invention, alternating ribonucleotides aremodified in both the antisense and the sense strands of the compound.

In particularly preferred embodiments of the invention, the antisensestrand is phophorylated at the 5′terminus, and may or may not bephophorylated at the 3′terminus;and the sense strand may or may not bephophorylated at the 5′terminus and at the 3′terminus.

In another embodiment of the compound of the invention, theribonucleotides at the 5′ and 3′ termini of the antisense strand aremodified in their sugar residues, and the ribonucleotides at the 5′ and3′ termini of the sense strand are unmodified in their sugar residues.

The invention further provides a vector capable of expressing any of theaforementioned oligoribonucleotides in unmodified form in a cell afterwhich appropriate modification may be made.

The invention also provides a composition comprising one or more of thecompounds of the invention in a carrier, preferably a pharmaceuticallyacceptable carrier.

The invention also provides a composition comprising a carrier and oneor more of the compounds of the invention in an amount effective todown-regulate expression in a cell of a human TGaseII gene, whichcompound comprises a sequence substantially complementary to theSequence of (N)_(x).

The invention also provides a method of down-regulating the expressionof a human TGaseII gene by at least 50% as compared to a controlcomprising contacting an mRNA transcript of the gene with one or more ofthe compounds of the invention.

In one embodiment the compound is down-regulating TGaseII, whereby thedown-regulation of TGaseII is selected from the group comprisingdown-regulation of TGaseII function (which may be examined by anenzymatic assay or a binding assay with a known interactor of the nativegene/polypeptide, inter alia), down-regulation of TGaseII protein (whichmay be examined by Western blotting, ELISA or immuno-precipitation,inter alia) and down-regulation of TGaseII mRNA expression (which may beexamined by Northern blotting, quantitative RT-PCR, in-situhybridisation or microarray hybridisation, inter alia).

The invention also provides a method of treating a patient sufferingfrom fibrosis or a fibrosis-related pathology, comprising administeringto the patient a composition of the invention in a therapeuticallyeffective dose so as to thereby treat the patient.

The invention also provides a method of treating a patient sufferingfrom a pathology related to aberrant cross-linking of cellular proteinsvia Transglutaminase II comprising administering to the patient acomposition of the invention in a therapeutically effective dose so asto thereby treat the patient.

The invention also provides a use of a therapeutically effective dose ofone or more compounds of the invention for the preparation of acomposition for promoting recovery in a patient suffering from fibrosisor a fibrosis-related pathology or a pathology related to aberrantcrosslinking of cellular proteins via Transglutaminase II.

Fibrotic diseases or diseases in which fibrosis is evident(fibrosis-related pathology) include both acute and chronic forms offibrosis of organs, including all etiological variants of the following:pulmonary fibrosis, including interstitial lung disease and fibroticlung disease, liver fibrosis, cardiac fibrosis including myocardialfibrosis, kidney fibrosis including chronic renal failure, skin fibrosisincluding scleroderma, keloids and hypertrophic scars; myelofibrosis(bone marrow fibrosis); all types of ocular scarring includingproliferative vitreoretinopathy (PVR) and scarring resulting fromsurgery to treat cataract or glaucoma; inflammatory bowel disease ofvariable etiology, macular degeneration, Grave's ophthalmopathy, druginduced ergotism, psoriasis, glioblastoma in Li-Fraumeni syndrome,sporadic glioblastoma, myleoid leukemia, acute myelogenious leukemia,myelodysplastic syndrome, myeloproferative syndrome, gynecologicalcancel-, Kaposi's sarcoma, Hansen's disease, and collagenous colitis .

The compounds of the invention may be used to treat many other diseasesand conditions apart from fibrotic diseases. Other indications may beocular diseases including cataract, cardiovascular diseases especiallycardiac hypertrophy, atherosclerosis/restenosis, neurological diseases,including polyglutamine diseases (such as Huntington's disease),spinobulbar muscular atrophy, dentatorubral-pallidoluysian atrophy andspinocerebellar ataxias (SCAs) 1, 2, 3, 6, 7 and 17, Alzheimer's diseaseand Parkinson's disease.

The compound may have homologs wherein up to two of the ribonucleotidesin each terminal region a base is altered; the terminal region refers tothe four terminal ribonucleotides e.g. refers to bases 1-4 and/or 16-19in a 19-mer sequence and to bases 1-4 and/or 18-21 in a 21-mer sequence.

The preferred oligonucleotides of the invention are the oligonucleotideslisted in Tables A, B and C, preferably the oligonucleotides targetinghuman cDNA. The most preferred oligonucleotides of the invention are theoligonucleotides having inhibitory activity as demonstrated in Table D,preferably oligonucleotides targeting human TGaseII cDNA.

The presently most preferred compound of the invention is a blunt-ended19-mer oligonucleotide, i.e. x=y=19 and Z and Z′ are both absent; theoligonucleotide is phosphorylated at the 5′position of the antisensestrand and at the 3′ position of the sense strand wherein alternatingribonucleotides are modified at the 2′ position in both the antisenseand the sense strands, wherein the moiety at the 2′ position is methoxy(2′-0-methyl) and wherein the ribonucleotides at the 5′ and 3′ terminiof the antisense strand are modified in their sugar residues, and theribonucleotides it the 5′ and 3′ termini of the sense strand areunmodified in their sugar residues. The presently most preferred suchcompound is TG_HMRG1. The antisense strand of TG_HMRG1 has SEQ ID NO: 22and the sense strand has SEQ ID NO: 4. Other preferred compounds areTG_HMG1 and TG_HM1, which have the antisense strand represented by SEQID NOS: 23 and 25 respectively and the sense strand represented by SEQID NO: 5 and 7 respectively.

In one aspect of the invention the oligonucleotide comprises adouble-stranded structure, whereby such double-stranded structurecomprises

-   -   a first strand and a second strand, whereby    -   the first strand comprises a first stretch of contiguous        nucleotides and the second strand comprises a second stretch of        contiguous nucleotides, whereby    -   the first stretch is either complementary or identical to a        nucleic acid sequence coding for TGaseII and whereby the second        stretch is either identical or complementary to a nucleic acid        sequence coding for TGaseII.

In an embodiment the first stretch and/or the second stretch comprisesfrom about 14 to 40 nucleotides, preferably about 18 to 30 nucleotides,more preferably from about 19 to 27 nucleotides and most preferably fromabout 19 to 23 nucleotides, in particular from about 19 to 21nucleotides. In such an aspect the oligonucleotide may be from 17-40nucleotides in length. Additionally, further nucleic acids according tothe present invention comprise at least 14 contiguous nucleotides of anyone of the SEQ. ID. NO.3 to SEQ ID NO: 416 and more preferably 14contiguous nucleotide base pairs at any end of the double-strandedstructure comprised of the first stretch and second stretch as describedabove.

The term “treatment” as used herein refers to administration of atherapeutic substance effective to ameliorate symptoms associated with adisease or condition, to lessen the severity or cure the disease orcondition, or to prevent the disease or condition from occurring.

In a particular embodiment, the administration comprises intravenousadministration. In another particular embodiment the administrationcomprises topical or local administration.

Additionally, the present invention provides a method of regulating apathology or disease (as recited above) in a patient in need of suchtreatment by administering to a patient a therapeutically effective doseof at least one antisense (AS) oligonucleotide or at least one siRNAagainst the nucleic acid sequences or a dominant negative peptidedirected against the TGaseII sequences or TGaseII proteins or anantibody directed against the TGaseII polypeptide.

Delivery: Delivery systems aimed specifically at the enhanced andimproved delivery of siRNA into mammalian cells have been developed,see, for example, Shen et al (FEBS letters 539: 111-114 (2003)), Xia etal., Nature Biotechnology 20: 1006-1010 (2002), Reich et al., MolecularVision 9: 210-216 (2003), Sorensen et al. (J.Mol.Biol. 327: 761-766(2003), Lewis et al., Nature Genetics 32: 107-108 (2002) and Simeoni etal., Nucleic Acids Research 31, 11: 2717-2724 (2003). siRNA has recentlybeen successfully used for inhibition in primates; for further detailssee Tolentino et al., Retina 24(1) February 2004 I 132-138.Respiratoryformulations for siRNA are described in U.S. patent application No.2004/0063654 of Davis et al. Cholesterol-conjugated siRNAs (and othersteroid and lipid conjugated siRNAs) can been used for delivery (seeSoutschek et al Nature 432: 173-177(2004) Therapeutic silencing of anendogenous gene by systemic administration of modified siRNAs; andLorenz et al. Bioorg. Med. Chemistry. Lett. 14:4975-4977 (2004) Steroidand lipid conjugates of siRNAs to enhance cellular uptake and genesilencing in liver cells.

The siRNAs or pharmaceutical compositions of the present invention areadministered and dosed in accordance with good medical practice, takinginto account the clinical condition of the individual patient, thedisease to be treated, the site and method of administration, schedulingof administration, patient age, sex, body weight and other factors knownto medical practitioners.

The “therapeutically effective dose” for purposes herein is thusdetermined by such considerations as are known in the art. The dose mustbe effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art. The compounds of thepresent invention can be administered by any of the conventional routesof administration. It should be noted that the compound can beadministered as the compound or as pharmaceutically acceptable salt andcan be administered alone or as an active ingredient in combination withpharmaceutically acceptable carriers, solvents, diluents, excipients,adjuvants and vehicles. The compounds can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, and intranasal administration as wellas intrathecal and infusion techniques. Implants of the compounds arealso useful. Liquid forms may be prepared for injection, the termincluding subcutaneous, transdermal, intravenous, intramuscular,intrathecal, and other parental routes of administration. The liquidcompositions include aqueous solutions, with and without organiccosolvents, aqueous or oil suspensions, emulsions with edible oils, aswell as similar pharmaceutical vehicles. In addition, under certaincircumstances the compositions for use in the novel treatments of thepresent invention may be formed as aerosols, for intranasal and likeadministration. The patient being treated is a warm-blooded animal and,in particular, mammals including man. The pharmaceutically acceptablecarriers, solvents, diluents, excipients, adjuvants and vehicles as wellas implant carriers generally refer to inert, non-toxic solid or liquidfillers, diluents or encapsulating material not reacting with the activeingredients of the invention and they include liposomes andmicrospheres. Examples of delivery systems useful in the presentinvention include U. S. Pat. Nos. 5,225,182; 5,169,383; 5,167,616;4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224;4,439,196; and 4,475,196. Many other such implants, delivery systems,and modules are well known to those skilled in the art. In one specificembodiment of this invention topical and transdermal formulations areparticularly preferred.

In general, the active dose of compound for humans is in the range offrom 1 ng/kg to about 20-100 mg/kg body weight per day, preferably about0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of onedose per day or twice or three or more times per day for a period of 1-4weeks or longer. Treatment for many years or even lifetime treatment isalso envisaged for some of the indications disclosed herein.

The present invention also provides for a process of preparing apharmaceutical composition, which comprises:

-   -   obtaining at least one double stranded siRNA compound of the        invention; and    -   admixing said compound with a pharmaceutically acceptable        carrier.

The present invention also provides for a process of preparing apharmaceutical composition, which comprises admixing a compound of thepresent invention with a pharmaceutically acceptable carrier.

In a preferred embodiment, the compound used in the preparation of apharmaceutical composition is admixed with a carrier in apharmaceutically effective dose. In a particular embodiment the compoundof the present invention is conjugated to a steroid or to a lipid or toanother suitable molecule e.g. to cholesterol.

Modifications or analogs of nucleotides can be introduced to improve thetherapeutic properties of the nucleotides. Improved properties includeincreased nuclease resistance and/or increased ability to permeate cellmembranes.

Accordingly, the present invention also includes all analogs of, ormodifications to, a oligonucleotide of the invention that does notsubstantially affect the function of the polynucleotide oroligonucleotide. In a preferred embodiment such modification is relatedto the base moiety of the nucleotide, to the sugar moiety of thenucleotide and/or to the phosphate moiety of the nucleotide.

In embodiments of the invention, the nucleotides can be selected fromnaturally occurring or synthetically modified bases. Naturally occurringbases include adenine, guanine, cytosine, thymine and uracil. Modifiedbases of the oligonucleotides include inosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl- adenines, 5-halouracil, 5-halo cytosine, 6-aza cytosine and 6-aza thymine, pseudouracil, 4-thiuracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine,8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substitutedadenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkylguanines, 8-hydroxyl guanine and other substituted guanines, other azaand deaza adenines, other aza and deaza guanines, 5-trifluoromethyluracil and 5-trifluoro cytosine.

In addition, analogs of nucleotides can be prepared wherein thestructures of the nucleotides are fundamentally altered and are bettersuited as therapeutic or experimental reagents. An example of anucleotide analog is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replacedwith a polyamide backbone similar to that found in peptides. PNA analogshave been shown to be resistant to degradation by enzymes and to haveextended lives in vivo and in vitro. Further, PNAs have been shown tobind more strongly to a complementary DNA sequence than to a DNAmolecule. This observation is attributed to the lack of charge repulsionbetween the PNA strand and the DNA strand. Other modifications that canbe made to oligonucleotides include polymer backbones, cyclic backbones,or acyclic backbones.

In one embodiment the modification is a modification of the phosphatemoiety, whereby the modified phosphate moiety is selected from the groupcomprising phosphothioate.

The compounds of the present invention can be synthesized by any of themethods that are well-known in the art for synthesis of ribonucleic (ordeoxyribonucleic) oligonucleotides. Such synthesis is, among others,described in Beaucage S. L. and Iyer R. P., Tetrahedron 1992; 48:2223-2311, Beaucage S. L. and Iyer R. P., Tetrahedron 1993; 49:6123-6194 and Caruthers M. H. et. al., Methods Enzymol. 1987; 154:287-313, the synthesis of thioates is, among others, described inEckstein F., Annu. Rev. Biochem. 1985; 54: 367-402, the synthesis of RNAmolecules is described in Sproat B., in Humana Press 2005 Edited byHerdewijn P.; Kap. 2: 17-31 and respective downstream processes are,among others, described in Pingoud A. et. al., in IRL Press 1989 Editedby Oliver R. W. A.; Kap. 7: 183-208 and Sproat B., in Humana Press 2005Edited by Herdewijn P.; Kap. 2: 17-31 (supra).

Other synthetic procedures are known in the art e.g. the procedures asdescribed in Usman et al., 1987,.J. Am. Chem. Soc., 109, 7845; Scaringeet al., 1990, Nucleic Acids Res., 18, 5433; Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684; and Wincott et al., 1997, Methods Mol.Bio., 74, 59, and these procedures may make use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. The modified (e.g. 2′-O-methylated)nucleotides and unmodified nucleotides are incorporated as desired.

The oligonucleotides of the present invention can be synthesizedseparately and joined together post-synthetically, for example, byligation (Moore et al., 1992 Science 256, 9923; Draper et al.,International PCT publication No. WO93/23569; Shabarova et al., 1991,Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides &Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

It is noted that a commercially available machine (available, interalia, from Applied Biosystems) can be used; the oligonucleotides areprepared according to the sequences disclosed herein. Overlapping pairsof chemically synthesized fragments can be ligated using methods wellknown in the art (e.g., see U.S. Pat. No. 6,121,426). The strands aresynthesized separately and then are annealed to each other in the tube.Then, the double-stranded siRNAs are separated from the single-strandedoligonucleotides that were not annealed (e.g. because of the excess ofone of them) by HPLC. In relation to the siRNAs or siRNA fragments ofthe present invention, two or more such sequences can be synthesized andlinked together for use in the present invention.

The compounds of the invention can also be synthesized via a tandemsynthesis methodology, as described in US patent application publicationNo. US2004/0019001 (McSwiggen), wherein both siRNA strands aresynthesized as a single contiguous oligonucleotide fragment or strandseparated by a cleavable linker which is subsequently cleaved to provideseparate siRNA fragments or strands that hybridize and permitpurification of the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker.

The compounds of the present invention can be delivered either directlyor with viral or non-viral vectors. When delivered directly thesequences are generally rendered nuclease resistant.

Alternatively the sequences can be incorporated into expressioncassettes or constructs such that the sequence is expressed in the cellas discussed herein below. Generally the construct contains the properregulatory sequence or promoter to allow the sequence to be expressed inthe targeted cell. Vectors optionally used for delivery of the compoundsof the present invention are commercially available, and may be modifiedfor the purpose of delivery of the compounds of the present invention bymethods known to one of skill in the art.

It is also envisaged that a long double stranded oligonucleotide(typically 25-500 nucleotides in length) comprising one or more of thesequences of the oligonucleotides of the invention may be delivered andmay be processed intracellularly by endogenous cellular complexes (e.g.by DICER as described above) to produce smaller double strandedoligonucleotides which are oligonucleotides of the invention.

As used herein, the term “polypeptide” refers to, in addition to apolypeptide, an oligopeptide, peptide and a full protein.

Animal model systems: Testing the active siRNAs of the invention may bedone in predictive animal models. Several models for kidney fibrosis aredescribed in Example 3.

Two models of liver fibrosis in rats are the Bile Duct Ligation (BDL)with sham operation as controls, and CCl₄ poisoning, with olive oil fedanimals as controls, as described in the following references:Lotersztajn S, Julien B, Teixeira-Clerc F, Grenard P, Mallat A, HepaticFibrosis: Molecular Mechanisms and Drug Targets. Annu Rev PharmacolToxicol. 2004 Oct. 07; Uchio K, Graham M, Dean N M, Rosenbaum J,Desmouliere A., Down-regulation of connective tissue growth factor andtype I collagen mRNA expression by connective tissue growth factorantisense oligonucleotide during experimental liver fibrosis. WoundRepair Regen. 2004 January-February; 12(1):60-6. ;and. Xu X Q, Leow C K,Lu X, Zhang X, Liu J S, Wong W H, Asperger A, Deininger S, EastwoodLeung H C., Molecular classification of liver cirrhosis in a rat modelby proteomics and bioinformatics Proteomics. 2004 October;4(10):3235-45.

Models for ocular scarring are well known in the art e.g. Sherwood M Bet al., J Glaucoma. 2004 October;13(5):407-12. A new model of glaucomafiltering surgery in the rat; Miller M H et al., Ophthalmic Surg. 1989May;20(5):350-7. Wound healing in an animal model of glaucomafistulizing surgery in the rabbit, vanBockxmeer F M et al., Retina. 1985Fall-Winter; 5(4): 239-52. Models for assessing scar tissue inhibitors;Wiedemann P et al., J Pharmacol Methods. 1984 August; 12(1): 69-78.Proliferative vitreoretinopathy: the rabbit cell injection model forscreening of antiproliferative drugs.

Models of cataract are described in the following publications: The roleof Src family kinases in cortical cataract formation. Zhou J, MenkoAS.Invest Ophthalmol Vis Sci. 2002 July;43(7):2293-300; Bioavailabilityand anticataract effects of a topical ocular drug delivery systemcontaining disulfiram and hydroxypropyl-beta-cyclodextrin onselenite-treated rats.Wang S, Li D, Ito Y, Nabekura T, Wang S, Zhang J,Wu C. Curr Eye Res. 2004 July;29(1):51-8; and Long-term organ culturesystem to study the effects of UV-Airradiation on lens transglutaminase.Weinreb O, Dovrat A.; Curr Eye Res. 2004 July;29(1):51-8.

Antibody Production

By the term “antibody” as used in the present invention is meant bothpoly- and mono-clonal complete antibodies as well as fragments thereof,such as Fab, F(ab′)2, and Fv, which are capable of binding the epitopicdeterminant. These antibody fragments retain the ability to selectivelybind with its antigen or receptor and are exemplified as follows, interalia:

-   (1) Fab, the fragment which contains a monovalent antigen-binding    fragment of an antibody molecule can be produced by digestion of    whole antibody with the enzyme papain to yield a light chain and a    portion of the heavy chain;-   (2) (Fab′)2, the fragment of the antibody that can be obtained by    treating whole antibody with the enzyme pepsin without subsequent    reduction; F(ab′2) is a dimer of two Fab fragments held together by    two disulfide bonds;-   (3) Fv, defined as a genetically engineered fragment containing the    variable region of the light chain and the variable region of the    heavy chain expressed as two chains; and-   (4) Single chain antibody (SCA), defined as a genetically engineered    molecule containing the variable region of the light chain and the    variable region of the heavy chain linked by a suitable polypeptide    linker as a genetically fused single chain molecule.

Such fragments having antibody functional activity can be prepared bymethods known to those skilled in the art (e.g. Bird et al. (1988)Science 242:423-426)

Conveniently, antibodies may be prepared against the immunogen orportion thereof, for example, a synthetic peptide based on the sequence,or prepared recombinantly by cloning techniques or the natural geneproduct and/or portions thereof may be isolated and used as theimmunogen. Immunogens can be used to produce antibodies by standardantibody production technology well known to those skilled in the art,as described generally in Harlow and Lane (1988), Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., and Borrebaeck (1992), Antibody Engineering—A Practical Guide, W.H. Freeman and Co., NY.

For producing polyclonal antibodies a host, such as a rabbit or goat, isimmunized with the immunogen or immunogen fragment, generally with anadjuvant and, if necessary, coupled to a carrier; antibodies to theimmunogen are collected from the sera. Further, the polyclonal antibodycan be absorbed such that it is monospecific; that is, the sera can beabsorbed against related immunogens so that no cross-reactive antibodiesremain in the sera, rendering it monospecific.

For producing monoclonal antibodies the technique involveshyperimmunization of an appropriate donor with the immunogen, generallya mouse, and isolation of splenic antibody-producing cells. These cellsare fused to an immortal cell, such as a myeloma cell, to provide afused cell hybrid that is immortal and secretes the required antibody.The cells are then cultured, in bulk, and the monoclonal antibodiesharvested from the culture media for use.

For producing recombinant antibody see generally Huston et al. (1991)“Protein engineering of single-chain Fv analogs and fusion proteins” inMethods in Enzymology (JJ Langone, ed., Academic Press, New York, N.Y.)203:46-88; Johnson and Bird (1991) “Construction of single-chain Fvbderivatives of monoclonal antibodies and their production in Escherichiacoli in Methods in Enzymology (JJ Langone, ed.; Academic Press, NewYork, N.Y.) 203:88-99; Mernaugh and Mernaugh (1995) “An overview ofphage-displayed recombinant antibodies” in Molecular Methods In PlantPathology (RP Singh and US Singh, eds.; CRC Press Inc., Boca Raton,Fla.:359-365). In particular scFv antibodies are described in WO2004/007553 (Tedesco and Marzari). Additionally, messenger RNAs fromantibody-producing B-lymphocytes of animals, or hybridoma can bereverse-transcribed to obtain complementary DNAs (cDNAs). Antibody cDNA,which can be full or partial length, is amplified and cloned into aphage or a plasmid. The cDNA can be a partial length of heavy and lightchain cDNA, separated or connected by a linker. The antibody, orantibody fragment, is expressed using a suitable expression system toobtain recombinant antibody. Antibody cDNA can also be obtained byscreening pertinent expression libraries.

The antibody can be bound to a solid support substrate or conjugatedwith a detectable moiety or be both bound and conjugated as is wellknown in the art. (For a general discussion of conjugation offluorescent or enzymatic moieties see Johnstone & Thorpe (1982.),Immunochemistry in Practice, Blackwell Scientific Publications, Oxford).The binding of antibodies to a solid support substrate is also wellknown in the art (for a general discussion, see Harlow & Lane (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPublications, New York; and Borrebaeck (1992), Antibody Engineering—APractical Guide, W. H. Freeman and Co.). The detectable moietiescontemplated with the present invention can include, but are not limitedto, fluorescent, metallic, enzymatic and radioactive markers such asbiotin, gold, ferritin, alkaline phosphatase, β-galactosidase,peroxidase, urease, fluorescein, rhodamine, tritium, ¹⁴C and iodination.

Additional compounds which are also considered to be useful in thetreatment of the diseases and disorders discussed herein may beantisense DNA molecules (which can be generated using the sequence inFIG. 1 by methods known in the art), catalytic RNAs such as ribozymes,polypeptides such as dominant negative peptides (which can be generatedusing the sequence in FIG. 2 by methods known in the art) or otherpolypeptide inhibitors. Antisense DNA molecules which comprise the siRNAsequences disclosed herein (with the appropriate nucleic acidmodifications stemming from the differences between DNA and RNA) areparticularly desirable and may be used in the same capacity as theircorresponding siRNAs for all uses and methods disclosed herein.

Screening of TGase Inactivating Compounds

Some of the compounds and compositions of the present invention may beused in a screening assay for identifying and isolating compounds thatmodulate the activity of TGaseII, in particular compounds that modulatefibrotic disease or fibrosis-related pathology. The compounds to bescreened comprise inter alia substances such as small chemicalmolecules, antibodies especially neutralizing antibodies, inhibitorypolypeptides and dominant negative peptides.

The inhibitory activity of the compounds of the present invention onTGaseII polypeptide enzymatic activity or binding of the compounds ofthe present invention to TGaseII may be used to determine theinteraction of an additional compound with the TGaseII polypeptide,e.g., if the additional compound competes with the antibodies ordominant-negative peptides of the present invention for TGaseIIinhibition, or if the additional compound rescues said inhibition. Theinhibition or activation can be tested by various means, such as, interalia, assaying for the product of the activity of the TGaseIIpolypeptide or displacement of binding compound from the TGaseIIpolypeptide in radioactive or fluorescent competition assays.

The present invention additionally provides for a process of obtaining acompound capable of inhibiting the enzymatic activity of a TGaseIIpolypeptide, preferably a human TGaseII polypeptide, that comprises thesteps of:

-   -   (i) contacting the TGaseII polypeptide or cells expressing the        TGaseII polypeptide with the compound, a lysyl donor and with a        glutamyl donor which is a biological molecule;    -   (ii) removing excess lysyl donor from the mixture of step (i);    -   (iii) measuring the amount of the lysyl-glutamyl crosslinked        material produced in the presence of the compound;    -   (iv) comparing the amount of lysyl-glutamyl crosslinked material        measured in step (iii) to that measured in the absence of the        compound under controlled conditions, wherein a decrease in the        amount of lysyl-glutamyl crosslinked material is indicative of        inhibition of the TGaseII polypeptide activity by the compound;        and    -   (v) identifying the compound as an inhibitor of TGaseII        polypeptide activity.

In one embodiment of all the processes described herein, the TGaseIIpolypeptide used in such process comprises consecutive amino acids, thesequence of which is set forth in SEQ ID NO: 2.

In another embodiment, the lysyl donor of step (i) is tagged and themeasurement in step (iii) is of the tag related read-out, and wherein, adecrease in the read-out level is indicative of inhibition of theTGaseII polypeptide activity by the compound.

The glutamyl donor which is a biological molecule may be e.g.fibronectin. or collagen.

In another embodiment, the lysyl donor used in such process is cadaverintagged with a dansyl fluorescent tag.

In another embodiment the lysyl donor used in such process is taggedwith biotin, and in a specific embodiment, the lysyl donor is biotincadaverine. Biotin is detected by streptavidin (or a modifiedstreptavidin such as nutravidin) conjugated to readouts known in the art(e.g. conjugated to horseradish peroxidase, wherein the amount ofhorseradish peroxidase is quantified using tetramethylbenzidine as asubstrate).

in yet another embodiment of the invention, the glutamyl donor in theprovided process may be an extra-cellular matrix protein or a cellularprotein. The extra cellular matrix protein is selected from the groupconsisting of: fibronectin, collagen, osteonectin, ECM-gel (e.g. SigmaE-1270), and the intracellular protein is selected inter alia from thegroup consisting of: RhoA, fialmin, spectrin, vimentin, HSP90, HSP60,(see Orru S, Caputo I, D'Amato A, Ruoppolo M, Esposito C Proteomicsidentification of acyl-acceptor and acyl-donor substrates fortransglutaminase in a human intestinal epithelial cell line.Implications for celiac disease. J Biol Chem. 2003 Aug.22;278(34):31766-73).

In a different embodiment, the compound is contacted with cellsexpressing the TGaseII polypeptide. The cells that may be used in suchprocess may either be separated or present in a tissue. The cells mayexpress the TGaseII polypeptide naturally or as a result of having beentransfected with TGaseII gene, either transiently or stably or mayover-express the TGaseII gene and the activity of TGaseII will becompared between over-expressors to normal TGaseII expressing cells. Ina further embodiment, the glutamyl donor is either a natural product ofthe cells or it is added from outside.

In a different embodiment, the TGaseII polypeptide is contacted with thecompound. The TGaseII polypeptide can be either immobilized or free in asolution. In an embodiment of the provided screening process theglutamyl donor is immobilized.

In a further embodiment, the processes provided for obtaining a compoundcapable of inhibiting the activity of human TGaseII may comprise afurther step in which prior to step (i) TGaseII is contacted with asecond compound known to bind TGaseII.

In one embodiment of the invention, the tag read-out in the providedprocesses described herein is the interaction of biotin with neutravidinconjugated to horseradish peroxidase, wherein the amount of cell-boundhorseradish peroxidase is quantified using TMB (tetramethylbenzidine) asa substrate.

In yet a further embodiment, the compound obtained by the providedprocess inhibits the activity of the TGaseII polypeptide at least 2-foldmore effectively than it inhibits the activity of at least one othermember of the TGase family, and more preferably the inhibition is atleast 10- fold, 50-fold and even 100-fold more effective. In suchprocess, either the TGaseII polypeptide or the second compound may beimmobilized.

In one another embodiment, the compound obtained by any of the providedprocesses is an antibody.

The invention further provides a process of obtaining a compound whichmodulates the activity of a TGaseII polypeptide, preferably a humanTGaseII polypeptide, which comprises the steps of:

-   -   (i) contacting the TGaseII polypeptide with an interactor with        which the TGaseII polypeptide interacts specifically in vivo;    -   (ii) contacting the TGaseII polypeptide or the interactor with        said compound; and    -   (iii) measuring the effect of the compound on the interaction        between TGaseII polypeptide and the interactor by measuring a        parameter related to fibrosis; and    -   (iv) comparing the effect measured in step (iii) with the effect        measured in the absence of the compound, a change in the effect        measured indicating that the compound modulates the activity of        the human TGaseII polypeptide.

In one embodiment of the invention the compound obtained by the providedprocess inhibits the activity of a human TGaseII polypeptide.

In another embodiment of the invention the TGaseII polypeptide in suchprocess comprises consecutive amino acids, the sequence of which is setforth in SEQ ID NO: 2. In a further embodiment either the TGaseIIpolypeptide or the interactor are immobilized.

In a further embodiment of the invention, the parameter measured in anyof the provided processes is related to a pathology characterized byTGaseII up-regulation in general and to fibrosis related pathologies asdefined above, and also to cataract, cardiovascular diseases,neurological disorders, polyglutamine diseases including Huntington'sdisease (HD), spinobulbar muscular atrophy, dentatorubral-pallidoluysianatrophy and spinocerebellar ataxias (SCAs) 1, 2, 3, 6, 7 and 17),Alzheimer's and Parkinson's disease, coeliac disease and osteoarthritis.It is another object of the present invention to provide a process ofobtaining a compound capable of inhibiting the activity of a humanTGaseII polypeptide that comprises the steps of:

-   -   (i) contacting the TGaseII polypeptide or cells expressing the        TGaseII polypeptide with a plurality of compounds, a tagged        lysyl donor and with a glutamyl donor which is a biological        molecule;    -   (ii) washing excess tagged lysyl donor from the mixture of step        (i); and    -   (iii) measuring the tag related read-out in the presence of the        plurality of compounds;    -   (iv) comparing the tag read-out measured in step (iii) to that        measured in the absence of the plurality of compounds under        controlled conditions, wherein a decrease in the read-out level        is indicative of inhibition of the TGaseII polypeptide activity        by the plurality of compounds;    -   (v) separately determining which compound or compounds present        in the plurality inhibit the activity of a human TGaseII.    -   It is another aspect of the present invention to provide a        cell-based process of obtaining a compound capable of inhibiting        the activity of a TGaseII polypeptide, preferably human TGaseII        polypeptide, that comprises the steps of:

-   (i) contacting cells expressing active TGaseII polypeptide with the    compound and with a lysyl donor;

-   (ii) removing excess lysyl donor from the mixture of step (i);

-   (iii) measuring the amount of lysyl-glutamyl crosslinked material    produced in the presence of the compound;

-   (iv) comparing the amount of lysyl-glutamyl crosslinked material    measured in step (iii) to that measured in the absence of the    compound under controlled conditions, wherein a decrease in the    amount of lysyl-glutamyl crosslinked material is indicative of    inhibition of the TGaseII polypeptide activity by the compound; and

-   (v) identifying the compound as an inhibitor of TGaseII polypeptide

It is another aspect of the present invention to provide a cell-basedprocess of obtaining a compound capable of inhibiting the activity of ahuman TGaseII polypeptide that comprises the steps of:

-   -   a. contacting cells expressing the TGaseII polypeptide with a        plurality of compounds and with a lysyl donor;    -   b. removing excess lysyl donor from the mixture of step (i); and    -   c. measuring the amount of the lysyl-glutamyl crosslinked        material produced in the presence of the plurality of compounds;    -   d. comparing the amount of lysyl-glutamyl crosslinked material        measured in step (iii) to that measured in the absence of the        plurality of compounds under controlled conditions, wherein a        decrease in the amount of lysyl-glutamyl crosslinked material is        indicative of inhibition of the TGaseII polypeptide activity by        the plurality of compounds; and    -   (e) separately determining which compound or compounds present        in the plurality inhibit the activity of a human TGaseII.        Methods        General Methods in Molecular Biology

Standard molecular biology techniques known in the art and notspecifically described were generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, New York (1989), and as in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989) and as inPerbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, NewYork (1988), and as in Watson et al., Recombinant DNA, ScientificAmerican Books, New York and in Birren et al (eds) Genome Analysis: ALaboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press,New York (1998) and methodology as set forth in U.S. Pat. Nos.4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 andincorporated herein by reference. Polymerase chain reaction (PCR) wascarried out generally as in PCR Protocols: A Guide To Methods AndApplications, Academic Press, San Diego, Calif. (1990). In situ (incell) PCR in combination with Flow Cytometry can be used for detectionof cells containing specific DNA and mRNA sequences (e.g. Testoni etal., 1996, Blood 87:3822.) Methods of performing RT-PCR are also wellknown in the art.

The present invention is illustrated in detail below with reference toExamples, but is not to be construed as being limited thereto.

EXAMPLES Example 1

Design of Active siRANA Compounds

Using proprietary algorithms and the known sequence of cDNA of TGaseII(SEQ ID NO:1), the sequences of many potential siRNAs were generated.These are shown in the Tables below. Note that the abbreviations forspecies are: H: human; M: mouse; R: rat; G: guinea pig; C: cow.

Table A shows 18 siRNAs that have so far been chemically synthesized andtested for activity (see Example 2). All these siRNAs are 19-mers. InTable A the sense strands of siRNAs 1-18 have SEQ ID NOS: 3-20respectively, and the antisense strands of siRNAs 1-18 have SEQ ID NOS:21-38 respectively.

Table B below shows 153 additional 19-mer siRNAs, which have beengenerated by the proprietary algorithms but not yet tested for activity.In Table B, the sense strands of siRNAs I-153 have SEQ ID NOS: 39-191,respectively, and the antisense strands of siRNAs 1-153 have SEQ ID NOS:192-344 respectively.

Table C below shows 36 additional 21-mer siRNAs that have been generatedby the proprietary algorithms. In Table C, the sense strands of siRNAs1-36 have SEQ ID NOS: 345-380 respectively, and the antisense strands ofsiRNAs 1-36 have SEQ ID NOS: 381-416, respectively. TABLE A NM_198951NM_009373 NM_198851 M19646.1 No. name Sense strand Antisense strandSpecies (human) (mouse) (RAT) (GP) 1 TG_HMR1 ACAAGAGCGAGAUGAUCUGCAGAUCAUCUCGCUCUUGU hum_mus_rat [1077- [1069- [1033- 1095] 1087] 1051] 2TG_HMRG1 AGAGCGAGAUGAUCUGGAA UUCCAGAUCAUCUCGCUCU hum_mus_rat_GP [1080-[1072- [1036- [1048- 1098] 1090] 1054] 1066] 3 TG_HMG1ACCCCAAGUUCCUGAAGAA UUCUUCAGGAACUUGGGGU hum_mus_GP [699-717] [691-709][667-685] 4 TG_HMG2 GCGAGAUGAUCUGGAACUU AAGUUCCAGAUCAUCUCGC hum_mus_GP[1083- [1075- [1051- 1101] 1093] 1069] 5 TG_HM1 CAAAUCCAUCAACCGUUCCGGAACGGUUGAUGGAUUUG hum_mus [1339- [1331- 1357] 1349] 6 TG_M1GAACAUCCAUGAGAAACUU AAGUUUCUCAUGGAUGUUC mus 2599-2617 7 TG_M2CGACCUAUGCCAAGAGAAA UUUCUCUUGGCAUAGGUCG mus 164-182 8 TG_M3GGUGUGAUUUGGAGAUUCA UGAAUCUCCAAAUCACACC mus 118-136 9 TG_M4CCAACCACCUGAACAAACU AGUUUGUUCAGGUGGUUGG mus_rat [1468- [1432- 1486]1450] 10 TG_M5 GAACAAACUGGCAGAGAAA UUUCUCUGCCAGUUUGUUC mus_rat [1478-[1442- 1496] 1460] 11 TG_M6 GCUCUGUCAAGUUCAUCAA UUGAUGAACUUGACAGAGCmus_rat 601-619 12 TG_M7 GAUCCCUACUCUGAGAACA UGUUCUCAGAGUAGGGAUC mus_rat1692-1637 13 TG_M8 CCAGAGUGGUGACCAACUA UAGUUGGUCACCACUCUGG mus_rat[976-994] [940-958] 14 TG_M9 GCAACAAGAGCGAGAUGAU AUCAUCUCGCUCUUGUUGCmus_rat [1066- [1030- 1084] 1048] 15 TG_M10 GCAACCUGCUCAUCGAGUAUACUCGAUGAGCAGGUUGC mus_rat [1018- [982- 1036] 1000] 16 TG_H1GUGACCUAACCACUUAGCA UGCUAAGUGGUUAGGUCAC hum 1773-1791 17 TG_H2GUAGUGACCUAACCACUUA UAAGUGGUUAGGUCACUAC hum 1770-1788 18 TG_HG1GACGCUGGGACAACAACUA UAGUUGUUGUCCCAGCGUC hum_GP [816-834] [784-802]

TABLE B Guinea- human GI Mouse GI Rat GI pig GI Cow GI No. Method SensesiRNA AntiSense siRNA 39777598 31543859 42476286 387604 31343554 species1 Cross Sp UUCCACUGCUGGGUGGAGU ACUCCACCCAGCAGUGGAA 1100- 1092- 1056-1068- 1046- hmrgc 1118 1110 1074 1086 1064 2 Cross SpAAGAGCGAGAUGAUCUGGA UCCAGAUCAUCUCGCUCUU 1079- 1071- 1035- 1047- 1025-hmrgc 1097 1089 1053 1065 1043 3 Cross Sp CAAGAGCGAGAUGAUCUGGCCAGAUCAUCUCGCUCUUG 1078- 1070- 1034- 1046- 1024- hmrgc 1096 1088 10521064 1042 4 Cross Sp AACCACCUGAACAAACUGG CCAGUUUGUUCAGGUGGUU 1478- 1470-1434- — 1424- hmr 1496 1488 1452 1442 5 Cross Sp ACCACCUGAACAAACUGGCGCCAGUUUGUUCAGGUGGU 1479- 1471- 1435- — 1425- hmr 1497 1489 1453 1442 6Cross Sp CUGGAACUUCCACUGCUGG CCAGCAGUGGAAGUUCCAG 1093- 1085- 1049- 1061-1039- hm 1111 1103 1067 1079 1057 7 Cross Sp AACUUCCACUGCUGGGUGGCCACCCAGCAGUGGAAGUU 1097- 1089- 1056- 1065- 1043- hm 1115 1107 1071 10831061 8 Cross Sp AGGAGAAGAGCGAAGGGAC GUCCCUUCGCUCUUCUCCU 1185- 1177-1141- — 1131- hm 1203 1195 1156 1149 9 Cross Sp UGAUCUGGAACUUCCACUGCAGUGGAAGUUCCAGAUCA 1089- 1081- — 1057- 1035- hm 1107 1099 1075 1053 10Cross Sp GAGCGAGAUGAUCUGGAAC GUUCCAGAUCAUCUCGCUC 1081- 1073- 1037- 1049-1027- hm 1099 1091 1054 1067 1045 11 Cross Sp UCUGGAACUUCCACUGCUGCAGCAGUGGAAGUUCCAGA 1092- 1084- 1048- 1060- 1038- hm 1110 1102 1066 10781056 12 Cross Sp GAUCUGGAACUUCCACUGC GCAGUGGAAGUUCCAGAUC 1090- 1082- —1058- 1036- hm 1108 1100 1076 1054 13 Cross Sp GAGAUGAUCUGGAACUUCCGGAAGUUCCAGAUCAUCUC 1085- 1077- 1041- 1053- 1031- hm 1103 1095 1059 10711049 14 Cross Sp AGCGAGAUGAUCUGGAACU AGUUCCAGAUCAUCUCGCU 1082- 1074-1038- 1050- 1028- hm 1100 1092 1054 1068 1046 15 Cross SpAGAUGAUCUGGAACUUCCA UGGAAGUUCCAGAUCAUCU 1086- 1078- 1042- 1054- 1032- hm1104 1096 1060 1072 1050 16 Cross Sp CCCCAAGUUCCUGAAGAACGUUCUUCAGGAACUUGGGC 700-718 692-710 656-671 668-685 646-659 hm 17 CrossSp AUCUGGAACUUCCACUGCU AGCAGUGGAAGUUCCAGAU 1091- 1083- — 1059- 1037- hm1109 1101 1077 1055 18 Cross Sp AGCUUUGUGCUGGGCCACU AGUGGCCCAGCACAAAGCU485-503 477-495 — — — hm 19 Cross Sp AACCCCAAGUUCCUGAAGAUCUUCAGGAACUUGGGGUU 698-716 690-708 654-671 666-684 644-659 hm 20 CrossSp CAGGAGAAGAGCGAAGGGA UCCCUUCGCUCUUCUCCUG 1184- 1176- 1140- 1152- 1130-hm 1202 1194 1156 1170 1148 21 Cross Sp UUGUGCUGGGCCACUUCAUAUGAAGUGGCCCAGCACAA 489-507 481-499 — 462-475 — hm 22 Cross SpGAACUUCCACUGCUGGGUG CACCCAGCAGUGGAAGUUC 1096- 1088- 1056- 1064- 1042- hm1114 1106 1070 1082 1060 23 Cross Sp GGAACUUCCACUGCUGGGUACCCAGCAGUGGAAGUUCC 1095- 1087- 1051- 1063- 1041- hm 1113 1105 1069 10811059 24 Cross Sp ACUUCCACUGCUGGGUGGA UCCACCCAGCAGUGGAAGU 1098- 1090-1056- 1066- 1044- hm 1116 1108 1072 1084 1062 25 Cross SpCGAGAUGAUCUGGAACUUC GAAGUUCCAGAUCAUCUCG 1084- 1076- 1040- 1052- 1030- hm1102 1094 1054 1070 1048 26 Cross Sp AUGAUCUGGAACUUCCACUAGUGGAAGUUCCAGAUCAU 1088- 1080- 1044- 1056- 1034- hm 1106 1098 1062 10741052 27 Cross Sp UUUGUGCUGGGCCACUUCA UGAAGUGGCCCAGCACAAA 488-506 480-498— 462-474 — hm 28 Cross Sp GAUGAUCUGGAACUUCCAC GUGGAAGUUCCAGAUCAUC 1087-1079- 1043- 1055- 1033- hm 1105 1097 1061 1073 1051 29 Cross SpUGGAAGUUCCACUGCUGGG CCCAGCAGUGGAAGUUCCA 1094- 1086- 1050- 1062- 1040- hm1112 1104 1068 1080 1058 30 Cross Sp CUUUGUGCUGGGCCACUUCGAAGUGGCCCAGCACAAAG 487-505 479-497 — 462-473 — hm 31 Cross SpGCUUUGUGCUGGGCCACUU AAGUGGCCCAGCACAAAGC 486-504 478-496 — 462-472 — hm32 Cross Sp UCAGCACUAAGAGCGUGGG CCCACGCUCUUAGUGCUGA 1377- 1369- 1333- —— hr 1395 1387 1351 33 Cross Sp AGAUCAGCACUAAGAGCGU ACGCUCUUAGUGCUGAUCU1374- 1366- 1330- — — hr 1392 1381 1348 34 Cross Sp GAAGAUCAGCACUAAGAGCGCUCUUAGUGCUGAUCUUC 1372- 1364- 1328- — 1318- hr 1390 1381 1346 1329 35Cross Sp GAUCAGCACUAAGAGCGUG CACGCUCUUAGUGCUGAUC 1375- 1367- 1331- — —hr 1393 1381 1349 36 Cross Sp AGUAUGGCCAGUGCUGGGU ACCCAGCACUGGCCAUACU918-936 918-928 874-892 886-904 864-882 hr 37 Cross SpAAGUAUGGCCAGUGCUGGG CCCAGCACUGGCCAUACUU 917-935 — 873-891 885-903863-881 hr 38 Cross Sp AUCAGCACUAAGAGCGUGG CCACGCUCUUAGUGCUGAU 1376-1368- 1332- — — hr 1394 1386 1350 39 Cross Sp AAGAUCAGCACUAAGAGCGCCCUCUUAGUGCUGAUCUU 1373- 1365- 1329- — 1319- hr 1391 1381 1347 1329 40Cross Sp GGAUCCUAGACAUCUGCCU AGGCAGAUGUCUAGGAUCC 663-681 — — — 609-627hc 41 Cross Sp CGCUGGGACAACAACUACG CGUAGUUGUUGUCCCAGCG 818-836 810-826774-787 786-803 764-782 hc 42 Cross Sp CUUGGAAUUUUGGGCAGUUAACUGCCCAAAAUUCCAAG 636-654 628-646 592-610 — 582-600 hc 43 Cross SpGUCAACUGCAACGAUGACC GGUCAUCGUUGCAGUUGAC 782-800 774-792 738-756 750-760728-746 hc 44 Cross Sp CAAGAACAUACCUUGGAAU AUUCCAAGGUAUGUUCUUG 625-643 —— 599-610 571-589 hc 45 Cross Sp CUCAUCGAGUACUUCCGCA UGCGGAAGUACUCGAUGAG1034- 1026- 990- 1002- 980-998 hc 1052 1042 1006 1018 46 Cross SpGCAUGGUCAACUGCAACGA UCGUUGCAGUUGACCAUGC 777-795 770-784 733-748 746-760723-741 hc 47 Cross Sp CGGAUGCUGUGUACCUGGA UCCAGGUACACAGCAUCCG 534-552 —— — 480-498 hc 48 Cross Sp CCAUGACCAGAACAGCAAC GUUGCUGUUCUGGUCAUGG 1012-1008- 972-986 984-998 958-976 hc 1030 1022 49 Cross SpACAUACCUUGGAAUUUUGG CCAAAAUUCCAAGGUAUGU 630-648 — — 599-610 576-594 hc50 Cross Sp UCGAGUACUUCCGCAAUGA UCAUUGCGGAAGUACUCGA 1038- 1030- 994-1006- 984- hc 1056 1048 1006 1018 1002 51 Cross Sp AUGGUCAACUGCAACGAUGCAUCGUUGCAGUUGACCAU 779-797 771-789 735-753 747-760 725-743 hc 52 CrossSp ACCUUGGAAUUUUGGGCAG CUGCCCAAAAUUCCAAGGU 634-652 — — — 580-598 hc 53Cross Sp CAAGUUCAUCAAGAACAUA UAUGUUCUUGAUGAACUUG 616-634 608-621 572-585584-595 562-580 hc 54 Cross Sp UUCAUCAAGAACAUACCUU AAGGUAUGUUCUUGAUGAA620-638 — — — 566-584 hc 55 Cross Sp AUACCUUGGAAUUUUGGGCGCCCAAAAUUCCAAGGUAU 632-650 — — 600-610 578-596 hc 56 Cross SpAGUACUUCCGCAAUGAGUU AACUCAUUGCGGAAGUACU 1041- — — — 987- hc 1059 1005 57Cross Sp UGGGUGGAGUCGUGGAUGA UCAUCCACGACUCCACCCA 1109- 1101- 1065- 1077-1055- hc 1127 1119 1083 1095 1073 58 Cross Sp CCCAUGACCAGAACAGCAAUUGCUGUUCUGGUCAUGGG 1011- 1003- 967-985 979-997 957-975 hc 1029 1021 59Cross Sp CCAAGUUCAUCAAGAACAU AUGUUCUUCAUGAACUUGG 615-633 608-621 572-585583-595 561-579 hc 60 Cross Sp GUCAACCCCAAGUUCCUGA UCAGGAACUUGGGGUUGAC695-713 690-705 654-669 665-681 641-659 hc 61 Cross SpAUCAAGAACAUACCUUGGA UCCAAGGUAUGUUCUUGAU 623-641 — — 599-609 569-587 hc62 Cross Sp UGGUCAACUGCAACGAUGA UCAUGGUUGCAGUUGACCA 780-798 772-790736-754 748-760 726-744 hc 63 Cross Sp CAUGGUCAACUGCAACGAUAUCGUUGCAGUUGACCAUG 778-796 770-784 734-748 746-760 724-742 hc 64 CrossSp UGGAAUUUUGGGCAGUUUG CAAACUGCCCAAAAUUCCA 638-656 630-646 594-612 —584-602 hc 65 Cross Sp ACUGCAACGAUGACCAGGG CCCUGGUCAUCGUUGCAGU 786-804778-796 742-760 — 732-750 hc 66 Cross Sp AUCCUAGACAUCUGCCUGAUCAGGCAGAUGUCUAGGAU 665-683 — — — 611-629 hc 67 Cross SpCAACUGCAACGAUGACCAG CUGGUCAUCGUUGCAGUUG 784-802 — — — 730-748 hc 68Cross Sp GGAAUUUUGGGCAGUUUGA UCAAACUGCCCAAAAUUCC 639-657 631-646 595-613615-625 585-603 hc 69 Cross Sp AGCGGAUGCUGUGUACCUG CAGGUACACAGCAUCCGCU532-550 — — 500-511 478-496 hc 70 Cross Sp UUUGCCCACAUCACCAACAUGUUGGUGAUGUGGGCAAA 1574- 1566- 1530- — 1520- hc 1592 1577 1541 1538 71Cross Sp AAGAACAUACCUUGGAAUU AAUUCCAAGGUAUGUUCUU 626-644 — — 599-610572-590 hc 72 Cross Sp UCCUAGACAUCUGCCUGAU AUCAGGCAGAUGUCUAGGA 666-684 —— — 612-630 hc 73 Cross Sp CGAGUACUUCCGCAAUGAG CUCAUUGCGGAAGUACUCG 1039-1031- 995- 1007- 985- hc 1057 1049 1006 1018 1003 74 Cross SpUGUCAACCCCAAGUUCCUG CAGGAACUUGGGGUUGACA 694-712 690-704 654-668 665-680640-658 hc 75 Cross Sp GACGUCUUUGCCCACAUCA UGAUGUGGGCAAAGACGUC 1568-1560- 1524- — 1514- hc 1586 1577 1541 1532 76 Cross SpGAUGCUGUGUACCUGGACU AGUCCAGGUACACACCAUC 536-554 — — —482-500 hc 77 CrossSp AGAACAUACCUUGGAAUUU AAAUUCCAAGGUAUGUUCU 627-645 — — 599-610 573-591hc 78 Cross Sp ACGUCUUUGCCCACAUCAC GUGAUGUGGGCAAAGACGU 1569- 1561- 1525-— 1515- hc 1587 1577 1541 1533 79 Cross Sp AUCGAGUACUUCCGCAAUGCAUUGCGGAAGUACUCGAU 1037- 1029- 993- 1005- 983- hc 1055 1047 1006 10181001 80 Cross Sp AAGUUCAUCAAGAACAUAC GUAUGUUCUUGAUGAACUU 617-635 609-621573-585 585-595 563-581 hc 81 Cross Sp UGGAGUCGUGGAUGACCAGCUGGUCAUCCACGACUCCA 1113- 1105- 1069- 1081- 1059- hc 1131 1123 1087 10991077 82 Cross Sp GUGACAAGAGCGAGAUGAU AUCAUCUCGCUCUUGUCAC 1074- 1069-1033- 1045- 1020- hc 1092 1084 1048 1060 1038 83 Cross SpUCAUCGAGUACUUCCGCAA UUGCGGAAGUACUCGAUGA 1035- 1027- 991- 1003- 981-999hc 1053 1042 1006 1018 84 Cross Sp GUUCGUGCCAUCAAGGAGGCCUCCUUGAUGGCACGAAC 1226- 1224- 1188- 1194- 1172- hc 1244 1236 1200 12121190 85 Cross Sp CAUCAAGAACAUACCUUGG CCAAGGUAUGUUCUUGAUG 622-640 — — —568-586 hc 86 Cross Sp UACCUUGGAAUUUUGGGCA UGCCCAAAAUUCCAAGGUA 633-651 —— — 579-597 hc 87 Cross Sp GACAAGAGCGAGAUGAUCU AGAUCAUCUCGCUCUUGUC 1076-1069- 1033- 1045- 1022- hc 1094 1086 1050 1062 1040 88 Cross SpCGUCUUUGCCCACAUCACG GGUGAUGUGGGCAAAGACG 1570- 1566- 1530- — 1516- hc1588 1577 1541 1534 89 Cross Sp AUGCUGUGUACCUGGACUC GAGUCCAGGUACACAGCAU537-555 — — — 483-501 hc 90 Cross Sp UUGGAAUUUUGGGCAGUUUAAACUGCCCAAAAUUCCAA 637-655 629-646 593-611 — 583-601 hc 91 Cross SpGCCAAGUUCAUCAAGAACA UGUUCUUGAUGAACUUGGC 614-632 608-621 572-585 582-595560-578 hc 92 Cross Sp AGUUCAUCAAGAACAUACC GGUAUGUUCUUGAUGAACU 618-636610-621 574-585 — 564-582 hc 93 Cross Sp GUCUUUGCCCACAUCACCAUGGUGAUGUGGGCAAAGAC 1571- 1566- 1530- — 1517- hc 1589 1577 1541 1535 94Cross Sp GGGAUCCUAGACAUCUGCC GGCAGAUGUCUAGGAUCCC 662-680 — — — 608-626hc 95 Cross Sp GAUCCUAGACAUCUGCCUG CAGGCAGAUGUCUAGGAUC 664-682 — — —610-628 hc 96 Cross Sp AUGUCAACCCCAAGUUCCU AGGAACUUGGGGUUGACAU 693-711690-703 649-667 665-679 639-657 hc 97 Cross Sp UCAUCAAGAACAUACCUUGCAAGGUAUGUUCUUGAUGA 621-639 — — — 567-585 hc 98 Cross SpUCAAGAACAUACCUUGGAA UUCCAAGGUAUGUUCUUGA 624-642 — — 599-610 570-588 hc99 Cross Sp CAUCGAGUACUUCCGCAAU AUUGCGGAAGUACUCGAUG 1036- 1028- 992-1004- 982- hc 1054 1042 1005 1018 1000 100 Cross Sp GGAUGCUGUGUACCUGGACGUCCAGGUACACAGGAUCC 535-553 — — — 481-499 hc 101 Cross SpGAUGUCAACCCCAAGUUCC GGAACUUGGGGUUGACAUC 692-710 690-702 648-666 665-678638-656 hc 102 Cross Sp CAUGACCAGAACAGCAACC GGUUGCUGUUCUGGUCAUG 1013-1008- 972-987 984-999 959-977 hc 1031 1023 103 Cross SpGCCCAUGACCAGAACAGCA UGCUGUUCUGGUCAUGGGC 1010- 1002- 966-984 978-996956-974 hc 1028 1020 104 Cross Sp GAACAUACCUUGGAAUUUUAAAAUUCCAAGGUAUGUUC 628-646 — — 599-610 574-592 hc 105 Cross SpGUGGAGUCGUGGAUGACCA UGGUCAUCCACGACUCCAC 1112- — — 1080- 1058- hc 11301098 1076 106 Cross Sp UCAACUGCAACGAUGACCA UGGUCAUCGUUGCAGUUGA 783-801 —— — 729-747 hc 107 Cross Sp AUUUUGGGCAGUUUGAAGA UCUUCAAACUGCCCAAAAU642-660 636-646 600-613 615-628 588-606 hc 108 Cross SpAACAUACCUUGGAAUUUUG CAAAAUUCCAAGGUAUGUU 629-647 — — 599-610 575-593 hc109 Cross Sp UGACAAGAGCGAGAUGAUC GAUCAUCUCGCUCUUGUCA 1075- 1069- 1033-1045- 1021- hc 1093 1085 1049 1061 1039 110 Cross Sp UUCGUGCCAUCAAGGAGGGCCCUCCUUGAUGGCACGAA 1227- 1224- 1188- 1195- 1173- hc 1245 1237 1201 12131191 111 Cross Sp UCAGCCCCAUGUCCUGGAU AUCCAGGACAUGGGGCUGA 846-864 — —814-832 792-810 hc 112 Cross Sp GGUCAACUGCAACGAUGAC GUCAUCGUUGCAGUUGACC781-799 773-791 737-755 749-760 727-745 hc 113 Cross SpAAUUUUGGGCAGUUUGAAG CUUCAAACUGCCCAAAAUU 641-659 636-646 600-613 615-627587-605 hc 114 Cross Sp CAGCGGAUGCUGUGUACCU AGGUACACAGCAUCCGCUG 531-549— — 499-511 477-495 hc 115 Cross Sp AACUGCAACGAUGACCAGGCCUGGUCAUCGUUGCAGUU 785-803 — — — 731-749 hc 116 Cross SpAUGACCAGAACAGCAACCU AGGUUGCUGUUCUGGUCAU 1014- 1008- 972-988 984- 960-978hc 1032 1024 1000 117 Cross Sp CAUACCUUGGAAUUUUGGG CCCAAAAUUCCAAGGUAUG631-649 — — 599-610 577-595 hc 118 Cross Sp CUUUGCCCACAUCACCAACGUUGGUGAUGUGGGCAAAG 1573- 1566- 1530- — 1519- hc 1591 1577 1541 1537 119Cross Sp GAGUACUUCCGCAAUGAGU ACUCAUUGCGGAAGUACUC 1040- 1032- 996- 1008-986- hc 1058 1050 1006 1018 1004 120 Cross Sp GGCAUGGUCAACUGCAACGCGUUGCAGUUGACCAUGCC 776-794 770-784 732-748 746-760 722-740 hc 121 CrossSp UCUUUGCCCACAUCACCAA UUGGUGAUGUGGGCAAAGA 1572- 1566- 1530- — 1518- hc1590 1577 1541 1536 122 Cross Sp GAAUUUUGGGCAGUUUGAA UUCAAACUGCCCAAAAUUC640-658 636-646 600-613 615-626 586-604 hc 123 Cross SpCCUUGGAAUUUUGGGCAGU ACUGCCCAAAAUUCCAAGG 635-653 — — — 581-599 hc 124Cross Sp GUUCAUCAAGAACAUACCU AGGUAUGUUCUUGAUGAAC 619-637 611-621 575-585— 565-583 hc 125 Cross Sp UGUAUCGCCUCAGCCUGGA UCCAGGCUGAGGCGAUACA441-459 — 402-415 409-427 3571- hg 3582 126 Cross Sp GCUCGGCCAAGUUCAUCAAUUGAUGAACUUGGCCGAGC 609-627 608-619 572-583 577-595 555-573 hg 127 CrossSp GGCUCGGCCAAGUUCAUCA UGAUGAACUUGGCCGAGCC 608-626 608-618 572-582576-594 554-572 hg 128 Cross Sp GGACGCUGGGACAACAACU AGUUGUUGUCCCAGCGUCC815-833 810-825 774-787 783-801 764-779 hg 129 Cross SpGGCAGUUUGAAGAUGGGAU AUCCCAUCUUCAAACUGCC 648-666 — — 616-634 594-612 hg130 Cross Sp AGUUUGAAGAUGGGAUCCU AGGAUCCCAUCUUCAAACU 651-669 — 607-625619-637 — hg 131 Cross Sp CAGUUUGAAGAUGGGAUCC GGAUCCCAUCUUCAAACUG650-668 — — 618-636 596-614 hg 132 Cross Sp AGGGCUCGGCCAAGUUCAUAUGAACUUGGCCGAGCCCU 606-624 — — 574-592 552-570 hg 133 Cross SpGCAGUUUGAAGAUGGGAUC GAUCCCAUCUUCAAACUGC 649-667 — — 617-635 595-613 hg134 Cross Sp CAACCCCAAGUUCCUGAAG CUUCAGGAACUUGGGGUUG 697-715 690-707654-671 665-683 643-659 hg 135 Cross Sp GGGCAGUUUGAAGAUGGGAUCCCAUCUUCAAACUGCCC 647-665 — 603-621 615-633 593-611 hg 136 Cross SpACGCUGGGACAACAACUAC GUAGUUGUUGUCCCAGCGU 817-835 810-826 774-787 785-803764-781 hg 137 Cross Sp UACUGCUGUGGCCCAGUUC GAACUGGGCCACAGCAGUA 1205-1197- 1161- 1173- 1151- hg 1223 1213 1177 1191 1169 138 SingleUUGCCCACAUCACCAACAA UUGUUGGUGAUGUGGGCAA 1575- 1567- 1531- — 1521- h Sp1593 1577 1541 1538 139 Single CCAAGUACCUGCUCAACCU AGGUUGAGCAGGUACUUGG1677- — — 1660- — h Sp 1695 1672 140 Single CGGCCAAGUUCAUCAAGAAUUCUUGAUGAACUUGGCCG 612-630 608-621 572-585 580-595 560-576 h Sp 141Single CGAUGGGUCUGUGCACAAA UUUGUGCACAGACCCAUCG 1324- 1317- 1281- — — hSp 1342 1334 1298 142 Single CAGUGACUUUGACGUCUUU AAAGACGUCAAAGUCACUG1558- — 1517- 1535- — h Sp 1576 1528 1553 143 Single GCAUGAACAUGGGCAGUGAUCACUGCCCAUGUUCAUGC 1545- — — 1530-1540 — h Sp 1563 1540 144 SingleCAACUACGAGGCCAGUGUA UACACUGGCCUCGUAGUUG 244-262 239-250 — — — h Sp 145Single CAUGGGCAGUGACUUUGAC GUCAAAGUCACUGCCCAUG 1552- — — 1530- — h Sp1570 1547 146 Single GCCCACAUCACCAACAACA UGUUGUUGGUGAUGUGGGC 1577- — — —1523- h Sp 1595 1538 147 Single CGAACCACCUGAACAAACU AGUUUGUUCAGGUGGUUCG1476- 1470- 1434- — 1424- h Sp 1494 1486 1450 1440 148 SingleGGCCCGUUUUCCACUAAGA UCUUAGUGGAAAACGGGCC 322-340 — — — — h Sp 149 SingleUGACGUCUUUGCCCACAUC GAUGUGGGCAAAGACGUCA 1567- 1560- 1523- — 1514- h Sp1585 1577 1541 1531 150 Single GAACAUGGGCAGUGACUUU AAAGUCACUGCCCAUGUUC1549- — — 1530- — h Sp 1567 1544 151 Single GCAGUGACUUUGACGUCUUAAGACGUCAAAGUCACUGC 1557- — 1517- 1534- — h Sp 1575 1528 1552 152 SingleCUGUGUUCCUGGAGCAUUU AAAUGCUCCACGAACACAG 1724- — — — — h Sp 1742 153Single CAACCUGGAGCCUUUCUCU AGAGAAAGGCUCCAGGUUG 1696- — — 1105- — h Sp1714 1115

TABLE C Guinea- human mouse rat pig Cow No Method Sense siRNA AntiSensesiRNA 39777598 31543859 42476286 387604 31343554 file 1 Single SpCUAGACAUCUGCCUGAUCCUU AAGGAUCAGGCAGAUGUCUAG 668-688 — — — 614-630 hmrgc2 Single Sp CAACUGACAACAUGCUAGGUA UACCUAGCAUGUUGUCAGUUG 1752- — — — —hmrgc 1172 3 Single Sp CCAAGUUCAUCAAGAACAUAC GUAUGUUCUUGAUGAACUUGG615-635 608-621 572-585 583-595 561-581 hmrgc 4 Single SpCCUGAUCCUUCUAGAUGUCAA UUCACAUCUAGAAGGAUCAGG 679-699 — — — — hmrgc 5Cross Sp ACAAGAGCGAGAUGAUCUGGA UCCAGAUCAUCUCGCUCUUGU 1077- 1069- 1033-1045- 1023- hmrgc 1097 1089 1053 1065 1043 6 Single SpCAAGAACAUACCUUGGAAUUU AAAUUCCAAGGUAUGUUCUUG 625-645 — — 599-610 571-591hmrgc 7 Single Sp CGGCCAAGUUCAUCAAGAACA UGUUCUUGAUGAACUUGGCCG 612-632608-621 572-585 580-595 560-578 hmrgc 8 Single Sp CUGUGUUCCUGGAGCAUUUGUACAAAUGCUCCAGGAACACAG 1724- — — — — hmrgc 1744 9 Cross SpCAAGAGCGAGAUGAUCUGGAA UUCCAGAUCAUCUCGCUCUUG hmrgc 10 Single SpCAACUACGAGGCCAGUGUAGA UCUACACUGGCCUCGUAGUUG 244-264 239-250 — — — hmrgc11 Single Sp GGCCCGUUUUCCACUAAGAGA UCUCUUAGUGGAAAACGGGCC 322-342 — — — —hmrgc 12 Single Sp GCAACCUUCUCAUCGAGUACU AGUACUCGAUGAGAAGGUUGC 1026-1018- 982- 994- 972-992 hmrgc 1046 1038 1002 1014 13 Cross SpCGAGAUGAUCUGGAACUUCCA UGGAAGUUCCAGAUCAUCUCG 1084- 1076- 1040- 1052-1030- hm 1104 1096 1060 1072 1050 14 Cross Sp ACUUCCACUGCUGGGUGGAGUACUCCACCCAGCAGUGGAAGU 1098- 1090- 1056- 1066- 1044- hm 1118 1110 10741086 1064 15 Cross Sp AACCCCAAGUUCCUGAAGAAC GUUCUUCAGGAACUUGGGGUU698-718 690-710 654-671 666-685 644-659 hm 16 Cross SpAUGAUCUGGAACUUCCACUGC GCAGUGGAAGUUCCAGAUCAU 1088- 1080- 1044- 1056-1034- hm 1108 1100 1064 1076 1054 17 Cross Sp GAACUUCCACUGCUGGGUGGAUCCACCCAGCAGUGGAAGUUC 1096- 1088- 1056- 1064- 1042- hm 1116 1108 10721084 1062 18 Cross Sp UGGAACUUCCACUGCUGGGUG CACCCAGCAGUGGAAGUUCCA 1094-1086- 1050- 1062- 1040- hm 1114 1106 1070 1082 1060 19 Cross SpAGCGAGAUGAUCUGCAACUUC GAAGUUCCAGAUCAUCUCGCU 1082- 1074- 1038- 1050-1028- hm 1102 1094 1054 1070 1048 20 Cross Sp GAUGAUCUGGAACUUCCACUGCAGUGGAAGUUCCAGAUCAUC 1087- 1079- 1043- 1055- 1033- hm 1107 1099 10631075 1053 21 Cross Sp AGAGCGAGAUGAUCUGGAACU AGUUCCAGAUCAUCUCGCUCU 1080-1072- 1036- 1048- 1026- hm 1100 1092 1054 1068 1046 22 Cross SpCUGGAACUUCCACUGCUGGGU ACCCAGCAGUGGAAGUUCCAG 1093- 1085- 1049- 1061-1039- hm 1113 1105 1069 1081 1059 23 Cross Sp UGAUCUGGAACUUCCACUGCUAGCAGUGGAAGUUCCAGAUCA 1089- 1081- — 1057- 1035- hm 1109 1101 — 1077 105524 Cross Sp AAGAGCGAGAUGAUCUGGAAC GUUCCAGAUCAUCUCGCUCUU 1079- 1071-1035- 1047- 1025- hm 1099 1091 2054 1067 1045 25 Cross SpCAGCUUUGUGCUGGGCCACUU AAGUGGCCCAGCACAAAGCUG 484-504 476-496 — 452-472 —hm 26 Cross Sp GCUUUGUGCUGGGCCACUUCA UGAAGUGGCCCAGCACAAAGC 486-506478-498 — 462-474 — hm 27 Cross Sp AGAUGAUCUGGAACUUCCACUAGUGGAAGUUCCAGAUCAUCU 1086- 1078- 1042- 1054- 1032- hm 1106 1098 10621074 1052 28 Cross Sp CUUUGUGCUGGGCCACUUCAU AUGAAGUGGCCCAGCACAAAG487-507 479-499 — 462-475 — hm 29 Cross Sp GCGAGAUGAUCUGGAACUUCCGGAAGUUCCAGAUCAUCUCGC 1083- 1075- 1039- 1051- 1029- hm 1103 1095 10591071 1049 30 Cross Sp AUCUGGAACUUCCACUGCUGG CCAGCAGUGGAAGUUCCAGAU 1091-1083- 1047- 1059- 1037- hm 1111 1103 1067 1079 1057 31 Cross SpUCUGGAACUUCCACUGCUGGG CCCAGCAGUGGAAGUUCCAGA 1092- 1084- 1048- 1060-1038- hm 1112 1104 1068 1080 1058 32 Cross Sp GAUCUGGAACUUCCACUGCUGCAGCAGUGGAAGUUCCAGAUC 1090- 1082- 1046- 1058- 1036- hm 1110 1102 10661078 1056 33 Cross Sp GAGAUGAUCUGGAACUUCCAC GUGGAAGUUCCAGAUCAUCUC 1085-1077- 1041- 1053- 1031- hm 1105 1097 1061 1073 1051 34 Cross SpAGCUUUGUGCUGGGCCACUUC GAAGUGGCCCAGCACAAAGCU 485-505 477-497 — 453-473 —hm 35 Cross Sp GAGCGAGAUGAUCUGGAACUU AAGUUCCAGAUCAUCUCGCUC 1081- 1073-1037- 1049- 1027- hm 1101 1093 1054 1069 1047 36 Cross SpAACUUCCACUGCUGGGUGGAG CUCCACCCAGCAGUGGAAGUU 1097- 1089- 1056- 1065-1043- hm 1117 1109 1073 1085 1063

Example 2

Testing the siRNA Compounds for Anti-TGaseII Activity

1. Preparation of working solutions of siRNAs (double-strandedoligonucleotides)

Lyophilized oligonucleotides were dissolved in RNAse-freedouble-distilled water to produce a final concentration of 100 uM. Thediluted oligonucleotides were kept at room temperature for 15 min andimmediately frozen in liquid nitrogen. The oligonucleotides were storedat −80° C. and diluted before use with PBS.

II. Establishment of NRK49 cell line stably overexpressing rat TGaseII

The full length cDNA of rat TGaseII cloned in PLXSN vector (CLONTECH)was used for transfection into a Rat1 fibroblast cell line.Lipofectamine reagent (Invitrogen) was used as the transfection reagentusing the protocol described below. 48 hr following transfection,selection of stable clones was performed using G418 antibiotic(neomycin). The stable overexpression of TGaseII in this polyclonalpopulation was verified by Western blotting and TGaseII activity assay.The cells were designated as NRK49-TGaseII (TGASE-OE®).

III Activity assay for TGaseII in NRK49

4×10³NRK49-rat TGASE-OE cells were seeded per well in a 96 well plate.TGaseII activity was measured using a cell-based assay for TGaseIIactivity. Briefly, the growth medium was replaced with cell labelingmedium (DMEM in 0.1% BSA, 25 uM BC, 10 uM Calcimycin, 16.2 mM CaCl2) for45 min at 37° C. 45 min later, the cells were fixed with 4%formaldehyde, treated with 0.6% H₂O₂ and stained with Netravidin-HRP Abin order to test the cross-linking of biotin-labeled cadaverin. Theabsorbance was read at λ=450 nm. For normalization, the cells werestained with Hoechst dye solution. The activity of TGaseII is 75 foldhigher in NRK49-TGaseII cells compared to NRK49 control cells.

IV. Transfection by siRNA oligonucleotides using Lipofectamine2000reagent

2×10⁵ cells were seeded per well in 6 well-plates. After 24 hrs, thecells were transfected with TGaseII specific siRNA oligonucleotidesusing Lipofectamine2000 reagent (Invitrogen) according to the followingprocedure:

-   -   1. Before transfection, the cell medium was replaced with 1500        ul of fresh medium without antibiotics.    -   2. In a sterile plastic tube, Lipofectamine2000 reagent (the        amount is calculated according to 5 ul per well) was added to        250 ul of serum-free medium, and incubated for 5 min at room        temperature.    -   3. In another tube, the siRNA oligonucleotides (varying amounts        to fit the desired final concentration per well) were added to        250 ul of serum-free medium.

4. Lipofectamine2000 complex was combined with the siRNA solution andincubated for 20 min at room temperature.

-   -   5. The resulting mixture was added dropwise to the cells, and        the cells were incubated at 37° C. until analysis of siRNA        activity.

V. Testing the activity of anti TGaseII siRNAs in transfected human,mouse and rat cells

The activity of TGaseII siRNAs listed in Table A was tested in 3different cell lines originating from different species:

-   -   1. Mouse-specific TGaseII siRNAs were tested in mouse cells,        which express mouse TGaseII-NMUMG cells.    -   2. Human-specific TGaseII siRNA were tested in human HeLa cells        which express human TgaseII;see FIG. 3.    -   3. Rat-specific TGaseII siRNAs were tested in NRK49        overexpressing rat TGaseII (TGASE-OE® cells).

48 h to 6 days after transfection of siRNAs, the cells were harvestedand expression of TGaseII protein was examined by Western blot analysiswith specific antibodies. The decrease in TGaseII polypeptide expressionin the cells transfected with siRNA oligonucleotides specific to TGaseIIas compared to controls indicated that siRNAs elicited inhibitory effecton TGaseII protein expression, which can be quantified.

Table D below summarizes the information about TGaseII inhibitoryactivity of each siRNA oligonucleotide. The names of oligonucleotides inTable D correspond to the names used in Table A. Note that thecomparison is to the activity in each cell line transfected by differentsiRNAs, and not between cell lines. The presently most preferred humansiRNA is TG_HMRG1, which elicited high TGaseII inhibitory effect andalso inhibited mouse, rat and guinea pig TGaseII. Other preferred humansiRNAs are TG_HMG1 and TG_HM1. TABLE D Human Mouse Rat name Species TGIITGII TGII TG_HMR1 hum_mus_rat + − + TG_HMRG1 hum_mus_rat_GP ++++ +++++++ TG_HMG1 hum_mus_GP ++++ + ND TG_HMG2 hum_mus_GP ++++ ++++ ND(toxic) (toxic) TG_HM1 hum_mus ++++ + ND TG_M1 Mus + +++ ND TG_M2 Mus −+++ ND TG_M3 Mus +++ ++++ ND TG_M4 mus_rat ND ++ ++ TG_M5 mus_rat ++++++ +++ TG_M6 mus_rat ND ++ +++ TG_M7 mus_rat ND ++ ++++ TG_M8 mus_ratND ++ ++ TG_M9 mus_rat +++ +++ +++ TG_M10 mus_rat ND + +++ TG_H1 Hum −ND ND TG_H2 Hum − ND ND TG_HG1 hum_GP ++ ND ND

Example 3

Animal Models of Kidney Fibrosis

Testing of the in vivo therapeutic activity of anti-TGaseII siRNAs, orof neutralizing antibodies against TGaseII or of other TGaseIIinhibitors may be done in the following animal models of kidney fibrosisin which disease-associated overexpression of TGaseII was found (asdetected by in situ hybridization; see below).

A. Rat Diabetic and Aging Models of Kidney Fibrosis

Al. ZDF Rats

Kidney samples of 9-month-old ZDF rats (Zucker diabetic fatty rats)presented hydronephrotic changes with dilated calyces. Microscopicallythese samples displayed the features of glomerulosclerosis andtubulointerstitial fibrosis. In accordance with these morphologicalchanges, the expression of fibrosis marker genes (osteopontin (OPN),transforming growth factor β1 (TGF-β1) and procollagen α1(1) (Coll)), asmeasured by in situ hybridization, was significantly increased comparedto normal kidneys. Strong OPN expression was detectable in all tubularstructures in both kidney cortex and medulla. TGF-β1 expression waswidespread throughout interstitial cells. Some epithelial cells alsoshowed TGF-β1 expression. Coll expression was detectable by in situhybridization in most interstitial cells within the medulla, whilecortical expression was “focal”.

A2. Aged fa/fa (Obese Zucker) Rats

Fa/fa rats are are genetically deficient for leptin receptor; and, as aresult, develop insulin resistant diabetes (type II diabetes) withprogressive diabetic nephropathy, Kidney samples of 12-month-old fa/farats presented with high degree glomerulosclerosis and diffusetubulointerstitial fibrosis throughout both the cortex and the medulla.The pattern of fibrosis marker gene expression corresponded to themorphological changes. OPN was expressed in tubular structures in thecortex and in the medulla. Multiple interstitial cells expressed TGF-β1.Significantly, multiple foci and single interstitial cells showed strongColl expression in both cortex and medulla so that the number ofColl-expressing cells appeared to be higher in fa/fa kidney samples thanin ZDF samples.

A3. Aged Sprague-Dawley (SD) (Normal) Rats

Kidney samples of aged SD rats also showed increased accumulation ofcollagen in glomeruli and interstitial space and increased expression offibrosis marker genes. Significantly, the intensity of fibrotic changesvaried among samples. Thus, one of the four samples studied displayedvery few changes compared with young animals; in the second sample,fibrotic changes were confined only to “polar” regions, and theremaining two samples displayed uniform accumulation of collagen andelevated expression of marker genes throughout the sections

A4. Goto Kakizaki (GK)48-week-old Rats

GK rats are an inbred strain derived from Wistar rats, selected forspontaneous development of NIDDM (diabetes type II). Kidney samples fromboth GK and control Wistar 48-week-old rats showed accumulation ofcollagen in glomeruli and interstitial space. This accumulation was morepronounced in the GK samples.

TGaseII Hybridization Pattern

TGaseII-specific hybridization signal was associated with fibroticphenotype (diffuse or focal) in kidneys of both healthy and diabeticrats. Non-fibrotic kidney samples demonstrate a diffuse, low butdetectable TGaseII-specific hybridisation signal over proximal tubules.By 12 months of age, a stronger signal is concentrated over interstitialcells and epithelial lining of distended tubules that show clear signsof atrophy. In chronic renal failure sample (kidney from two years oldrat), a strong TGaseII expression is visible in tubular profiles linedby atrophic or proliferating epithelium cells within surroundinginterstitium. Similarily, ZDF samples showed intensification of thehybridization signal along with the progression of the fibrosis (oraging).

B. Induced Models of Kidney Fibrosis

B. Permanent Unilateral Ureteral Obstruction (UUO) Model

Unilateral ureteral obstruction (UUO) is a model of acute interstitialfibrosis occurring in healthy non-diabetic animals. Renal fibrosisdevelops within days following the obstruction.

To produce the model, rats were anaesthetized with Ketamin/Xylazine andthe abdominal cavity was opened. After exposure, the right ureter wasligated with a suture at the level of the lower kidney pole. Insham-operated rats, the ureter was exposed but not ligated. The studywas terminated at different timepoints following ureteral obstruction(e.g., at 24 hr, 5 days, 10 days, 15 days, 20 days and 25 days), and thekidneys were removed and examined.

Histological examination revealed that permanent UUO results in a rapidactivation of collagen synthesis by interstitial cells in both medullaand cortex. By 20-25 days of UUO, significant amounts of interstitialcollagen were deposited in the interstitial space, whereas glomerularaccumulation of collagen was confined to the outer capsule.

situ hybridization analysis of obstructed kidney samples indicated rapidand significant changes in the pattern of TGaseII expression dependenton the duration of UUO. After 24 hours of UUO, a very stronghybridization signal was detected over cortical tubular epithelium,whereas medullar epithelial cells showed much weaker signal. At 5 and 10days of UUO, the “foci” of strong hybridization signal were associatedwith the areas of developing tubulointerstitial fibrosis where thesignal was located to both epithelial and interstitial cells. Similarresults were observed in mice exposed to the same UUO model.

B2. 5/6 Nephrectomy

5/6 nephrectomy is another useful animal model for chronic renalinsufficiency (CRI) in which fibrosis is evident.

In summary, the results suggest that an increase in TGaseII expressionand activity level accompanies fibrotic changes in the kidneys.

Example 4

Establishment of TGaseII Role in Renal Fibrosis Employing the UUO ModelUsing TGaseII Knock-out Mice

TgaseII knock-out mice were obtained from Dr. Melino (De Laurenzi V,Melino G., Gene disruption of tissue transglutaminase, Mol Cell Biol.2001 January;21(1):148-55). The mice appeared phenotypically normal.Also no morphological changes were observed in kidneys obtained fromthese mice. TGaseII KO mice were then used for the analysis of thedevelopment of kidney fibrosis following induction of UUO by assessingkidney collagen accumulation compared to normal mice The results clearlydemonstrated that disruption of TGaseII results in statisticallysignificant reduced collagen accumulation following both three (3) andseven (7) days of obstruction, as compared to collagen accumulation inobstructed kidneys of wild type mice, thus implicating TGaseII as afunctionally important factor in kidney fibrosis. It is thus suggestedthat inhibition of TGaseII activity may have an anti-fibrotictherapeutic effect.

Example 5

Inhibition of TGaseII Activity Using Monoclonal Neutralizing Antibodies

Recombinant monoclonal human-anti-human antibodies against TGaseII wereobtained by screening of a phage display library. 720 clones werescreened for binding human TGaseII. Seven (7) clones were selected whichshowed intense binding to the enzyme. The activity assay demonstratedthat these antibodies inhibited the crosslinking activity of humanTGaseII enzyme at a range of concentrations (1-5 nM).

1. A compound having the structure:5′(N)_(x)−Z 3′ (antisense strand)3′Z′-(N′)_(y)5′ (sense strand)wherein each N and N′ is a ribonucleotidewhich may be modified or unmodified in its sugar residue and (N)_(x) and(N′)_(y) is an oligomer in which each consecutive N or N′ is joined tothe next N or N′ by a covalent bond; wherein each of x and y is aninteger between 19 and 40; wherein each of Z and Z′ may be present orabsent, but if present is dTdT and is covalently attached at the 3′terminus of the strand in which it is present; and wherein the sequenceof (N)_(x) comprises any one of the sequences set forth in SEQ ID NOS:21-38, 192-344 and 381-416.
 2. The compound of claim 1, wherein thecovalent bond is a phosphodiester bond.
 3. The compound of claim 2,wherein x=y.
 4. The compound of claim 3, wherein x=y=19.
 5. The compoundof claim 1 wherein Z and Z′ are both absent.
 6. The compound of claim 1wherein one of Z or Z′ is present.
 7. The compound of claim 1 whereinall of the ribonucleotides are unmodified in their sugar residues. 8.The compound of claim 1 wherein at least one ribonucleotide is modifiedin its sugar residue.
 9. The compound of claim 8, wherein themodification of the sugar residue comprises a modification at the 2′position.
 10. The compound of claim 9, wherein the modification at the2′ position results in the presence of a moiety selected from the groupcomprising amino, fluoro, methoxy, alkoxy and alkyl groups.
 11. Thecompound of claim 10, wherein the moiety at the 2′ position is methoxy(2′-0-methyl).
 12. The compound of claim 1 wherein alternatingribonucleotides are modified in both the antisense and the sensestrands.
 13. The compound of claim 1 wherein the ribonucleotides at the5′ and 3′ termini of the antisense strand are modified in their sugarresidues, and the ribonucleotides at the 5′ and 3′ termini of the sensestrand are unmodified in their sugar residues.
 14. The compound of claim1 wherein the antisense strand is phophorylated at the 5′ terminus, andmay or may not be phophorylated at the 3′ terminus; and wherein thesense strand may or may not be phophorylated at the 5′ terminus and atthe 3′ terminus.
 15. A vector capable of expressing the compound ofclaim
 1. 16. A composition comprising the compound of claim 1 in anamount effective to inhibit human TGaseII and a carrier.
 17. A method oftreating a patient suffering from a disorder comprising administering tothe patient an inhibitor of human TGaseII in a therapeutically effectivedose so as to thereby treat the patient.
 18. The method of claim 17,where the inhibitor is an siRNA.
 19. The method of claim 17, where theinhibitor is an antibody.
 20. (canceled)
 21. The method of claim 17,wherein the disorder is a fibrosis-related pathology.
 22. The method ofclaim 21, wherein the fibrosis-related pathology is kidney fibrosis,liver fibrosis, pulmonary fibrosis or ocular scarring.
 23. The method ofclaim 17, wherein the disorder is any one of an ocular diseaseespecially cataract, a cardiovascular disease especially cardiachypertrophy, atherosclerosis/restenosis, a neurological disease,including polyglutamine disease, spinobulbar muscular atrophy,dentatorubral-pallidoluysian atrophy, spinocerebellar ataxias (SCAs) 1,2, 3, 6, 7 and 17, Alzheimer's disease or Parkinson's disease.
 24. Acomposition comprising the vector of claim 15 in an amount effective toinhibit human TGaseII and a carrier.
 25. A method of treating a patientsuffering from a disorder comprising administering to the patient thecompounds of claim 1 in a therapeutically effective dose so as tothereby treat the patient.
 26. A method of treating a patient sufferingfrom a disorder comprising administering to the patient the vector ofclaim 15 in a therapeutically effective dose so as to thereby treat thepatient.